Tramdorins and methods of using tramdorins

ABSTRACT

A family of transmembrane domain proteins, the tramdorins, has been identified in human, mouse and rat. A variety of uses for these proteins is contemplated, including but not limited to peripheral myclination, the etiology of parathyroid adenomas, and the diagnosis and treatment of Central Nervous System (CNS) and non-CNS disorders.

[0001] This invention was made with government support under grantR01-NS40751, from the National Institute of Neurological Disorders andStroke. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to a gene family, the tramdorins.The genes may be used in a variety of applications, includingdiagnostics and therapeutics.

BACKGROUND

[0003] Identification of genes involved in nervous system function mayprovide markers for various steps in nervous system development and/orrepair following disease or injury. Some of these steps may also beimportant in diseases affecting the nervous system, including but notlimited to Charcot-Marie-Tooth syndrome, multiple sclerosis (MS) andGuillain-Barre syndrome, which are characterized by demyelination ofaxons in the peripheral or central nervous system. The frequencies ofthese diseases range from 1/1000 (U.S.) for multiple sclerosis to1.5/100,000 for Guillain-Barre syndrome. These diseases are devastatingfor those affected by them, often resulting in the loss of the use oflimbs. For example, Guillain-Barre syndrome generally requireshospitalization to provide respiratory and cardiac support during anepisode. In terms of economic costs, MS carries a substantial economicburden. A cost of illness (COI) study conducted by Bourdette et al.retrospectively examined costs to the US Veterans affairs for thetreatment of 165 patients with MS over a 3-year period [Bourdette D. N.et al., Arch Phys Med Rehabil 74:26-31 (1993)]. Drug costs were notincluded in the study. The average cost to the VA associated with thesepatients was estimated at $35,000 per year.

[0004] Gene products of genes involved in nervous system function mayalso localize in tissues not associated with the nervous system and alsomay provide markers for various steps in the development of tissues notassociated with the nervous system and/or repair of the same proximal todisease or injury.

[0005] What is needed are markers for various tissue processes(including but not limited to the nervous system) which can be used inthe development of diagnostic and therapeutic agents for a variety ofdisorders including, but not limited to, pathologies of the nervoussystem.

SUMMARY OF THE INVENTION

[0006] In several embodiments, isolated nucleic acid sequences encodingtramdorins are contemplated. In one embodiment, an isolated nucleic acidsequence selected from the group consisting of the cDNA sequenceencoding mouse tramd 2 (SEQ ID NO: 1), the cDNA sequence encoding mousetramd 3 (SEQ ID NO: 2), the cDNA sequence encoding human tramd 1 (SEQ IDNO: 3), the cDNA sequence encoding human tramd 2 (SEQ ID NO: 4), thecDNA sequence encoding human tramd 3 (SEQ ID NO: 5), the cDNA sequenceencoding rat tramd 1 (SEQ ID NO: 6), the genomic sequence of mouse tramd1 (SEQ ID NO: 7)and the genomic sequence of mouse tramd 3 (SEQ ID NO: 8)is contemplated.

[0007] In another embodiment, portions of nucleic acid sequences arecontemplated, including, but not limited to, a portion of the cDNAsequence encoding mouse tramd2 (SEQ ID NO: 1), a portion of the cDNAsequence encoding mouse tramd3 (SEQ ID NO: 2), a portion of the cDNAsequence encoding human tramd1 (SEQ ID NO: 3), a portion of the cDNAsequence encoding human tramd2 (SEQ ID NO: 4), a portion of the cDNAsequence encoding human tramd3 (SEQ ID NO: 5), a portion of the cDNAsequence encoding rat tramd1 (SEQ ID NO: 6), a portion of genomicsequence encoding mouse tramd1 (SEQ ID NO: 7) and a portion of thegenomic sequence encoding mouse tramd3 (SEQ ID NO: 8). Polymorphismswithin any of these sequences are also contemplated. Polymorphismswithin the genomic sequences of human tramd1 (SEQ ID NO: 9), humantramd2 (SEQ ID NO: 10), human tramd 3 (SEQ ID NO: 1 )and human tramdL(SEQ ID NO: 12) are also contemplated.

[0008] In some embodiments, said nucleic acid sequences or portions ofnucleic acid sequences may be fused to other nucleic acid sequences,such that gene fusions are provided.

[0009] In some embodiments, RNA transcribed from any of the sequences iscontemplated. The RNA transcripts may be synthesized in vivo, or invitro, by means known in the art. In some embodiments, RNA transcribedfrom the group consisting of mouse tramdorin 1 cDNA (SEQ ID NO: 13),mouse tramdorin 2 cDNA (SEQ ID NO: 1), mouse tramdorin 3 cDNA (SEQ IDNO: 2), human tramdorin 1 cDNA (SEQ ID NO: 3), human tramdorin 2 cDNA(SEQ ID NO: 4)and human tramdorin 3 cDNA (SEQ ID NO: 5) is contemplated.

[0010] In some embodiments, a vector comprising said isolated nucleicacid sequences (for example, human tramd 1 cDNA (SEQ ID NO: 3), humantramd2 cDNA (SEQ ID NO: 4), human tramd3 cDNA (SEQ ID NO: 5), mousetramd 1 cDNA (SEQ ID NO: 13), mouse tramd 2 cDNA (SEQ ID NO: 1), mousetramd 3 cDNA (SEQ ID NO: 2), or portions thereof) is contemplated. Insome embodiments, the vector is an expression vector. In someembodiments, the expression vector is suitable for expression of thenucleic acid sequences of interest in a eukaryotic host, while in otherembodiments, the expression vector is suitable for expression in aprokaryotic host. Representative eukaryotic hosts and host cells for theexpression vectors include, but are not limited to mouse, yeast, Xenopusand cultured mammalian cells.

[0011] In some embodiments, a host cell transfected by a vectorcomprising the nucleic acid sequences of interest is contemplated. Insome embodiments, the host cell is a eukaryotic cell. In someembodiments, the host cell is a Xenopus oocyte. In other embodiments,the host cell is a yeast cell. In yet other embodiments, the host cellis a mouse embryonic stem cell. Other host cells are contemplated,including but not limited to cultured mammalian cells (for example,COS-7 cells).

[0012] In some embodiments, a Xenopus oocyte is injected with nucleicacid comprising an RNA transcribed from a nucleic acid sequence ofinterest. In some embodiments, the RNA is a synthetic RNA and comprisesadditional flanking sequences and a cap.

[0013] In some embodiments, a method of identifying proteins whichinteract with tramdorins is contemplated. In one embodiment, the methodcomprises the yeast two-hybrid method. In one embodiment, the methodcomprises (a) providing: (i) a first recombinant vector comprising aportion of a tramdorin in operable combination with the DNA bindingdomain of a transcriptional activator, such that a chimeric protein willbe expressed, (ii) a population of second recombinant vectors, whereinsaid population comprises a library of cDNA sequences in operablecombination with the activation domain of a transcriptional activator,such that a population of chimeric proteins will be expressed, and (iii)a yeast host comprising a reporter gene in operable combination with theDNA binding sites for said transcriptional activator; (b) introducingsaid first vector and said population of second vectors into said yeasthost to generate a population of transformed yeast; (c) subjecting saidpopulation of transformed to yeast to conditions such that said chimericproteins are expressed; (d) screening said population for members whichexpress said reporter gene, and; (e) isolating the cDNA fragment fromsaid second vector from members of the population which express thereporter gene. In some embodiments, the portion of a tramdorin is aportion of mouse tramdorin 1 (SEQ ID NO: 14). In some embodiments, theportion of mouse tramdorin 1 may be the N-terminal 51 amino acids (SEQID NO: 15) or the first intracellular loop of tramdorin 1 (SEQ ID NO:16). In other embodiments, the portion of the tramdorin is a portion ofa tramdorin selected from the group consisting of mouse tramdorin 2 (SEQID NO: 17), mouse tramdorin 3 (SEQ ID NO: 18), human tramdorin 1 (SEQ IDNO: 19), human tramdorin 2 (SEQ ID NO: 20), human tramdorin 3 (SEQ IDNO: 21) and rat tramdorin 1 (SEQ ID NO: 22).

[0014] In other embodiments, a variation on the yeast two hybrid methodis contemplated to detect proteins which interact with tramdorins. Themethod comprises providing (i) a first recombinant vector comprising aportion of a tramdorin in operable combination with Sos, such that achimeric protein will be expressed, (ii) a population of secondrecombinant vectors, wherein said population comprises a library of cDNAsequences in operable combination with myristylation signal, such that apopulation of chimeric proteins will be expressed, and (iii) a yeasthost comprising a temperature sensitive mutation in the cdc25 gene; (b)introducing said first vector and said population of second vectors intosaid yeast host to generate a population of transformed yeast; (c)subjecting said population of transformed to yeast to conditions suchthat said chimeric proteins are expressed and said myristylation fusionswill be targeted to the membrane; (d) screening said population formembers which are viable at elevated temperatures, and; (e) isolatingthe cDNA fragment from said second vector from members of the populationwhich are viable at elevated temperatures. In some embodiments, saidtramdorin is a portion of mouse tramdorin 1 (SEQ ID NO: 14). In otherembodiments, said portion of tramdorin is selected from the groupconsisting of mouse tramdorin 2 (SEQ ID NO: 17), mouse tramdorin 3 (SEQID NO: 18), human tramdorin 1 (SEQ ID NO: 19), human tramdorin 2 (SEQ IDNO: 20), human tramdorin 3 (SEQ ID NO: 21) and rat tramdorin 1 (SEQ IDNO: 22).

[0015] In yet other embodiments, another variation on the yeasttwo-hybrid system is contemplated to identify proteins which interactwith tramdorins. In some embodiments, the method comprises (a) providing(i) a first recombinant vector comprising a full length tramdorin, (ii)a population of second recombinant vectors comprising a library of cDNAsequences fused to a ras gene lacking a myristylation signal, (iii) ayeast host comprising a temperature sensitive cdc25 mutation; (b)introducing said first recombinant vector and said population of secondrecombinant vectors into said yeast host to generate a population oftransformed yeast; (c) subjecting said population of transformed yeastto conditions such that said tramdorin protein is expressed andlocalizes to the membrane and said ras fusions are expressed; (d)screening said population for members which are viable at elevatedtemperatures, and; (e) isolating the cDNA fragment from said secondvector from members of the population which are viable at elevatedtemperatures. In some embodiments, said expressed tramdorin protein ismouse tramdorin 1 (SEQ ID NO: 14). In other embodiments, said tramdorinis selected from the group consisting of mouse tramdorin 2 (SEQ ID NO:17), mouse tramdorin 3 (SEQ ID NO: 18), human tramdorin1 (SEQ ID NO:19), human tramdorin 2 (SEQ ID NO: 20), human tramdorin 3 (SEQ ID NO:21) and rat tramdorin 1 (SEQ ID NO: 22).

[0016] In some embodiments, methods to detect tramdorin ligands arecontemplated. In one embodiment, the method comprises (a) providing (i)a recombinant expression vector comprising a tramdorin nucleic acidsequence, (ii) a host cell and (iii) labeled candidate ligand molecules;(b) introducing said recombinant expression vector into said host cellunder conditions such that said tramdorin nucleic acid sequence isexpressed; (c) contacting said tramdorin-expressing host cells with saidlabeled candidate ligand molecules, and; (d) measuring the relativeuptake of said labeled candidate ligand molecules into saidtramdorin-expressing host cells. In some embodiments, said host cellsare COS-7 cells. In other embodiments, said host cells are Xenopusoocytes. In some embodiments, the tramdorin nucleic acid sequence isselected from the group consisting of mouse tramdorin 1 cDNA (SEQ ID NO:13), mouse tramdorin 2 cDNA (SEQ ID NO: 1), mouse tramdorin 3 cDNA (SEQID NO: 2), human tramdorin 1 cDNA (SEQ ID NO: 3), human tramdorin 2 cDNA(SEQ ID NO: 4), human tramdorin 3 cDNA (SEQ ID NO: 5) and rat tramdorin1 cDNA (SEQ ID NO: 6). In some embodiments, said labeled candidateligand molecules are labeled with tritium [3H]. In some embodiments,said labeled candidate ligand molecules include, but are not limited to,small neutral amino acids and y-aminobutyric acid.

[0017] In some embodiments, methods to detect myelination arecontemplated. In such embodiments, the expression levels of tramdorin 1are measured in tissue or cells following injury or a disease process.In some embodiments, the tissue is damaged nervous tissue. In otherembodiments, the cells are Schwann cells co-cultured with neurons. Insome embodiments, tramdorin expression levels are measured by detectionof the levels of tramdorin 1 mRNA. Tramdorin 1 mRNA levels are detectedby Northern blot hybridization in some embodiments, while in otherembodiments, amplification of reverse transcribed mRNA (i.e.amplification of cDNA) is contemplated.

[0018] In some embodiments, methods to detect tramdL fusions inparathyroid tumors, including but not limited to parathyroid adenomasare contemplated. In some embodiments, the tramdL fusion is a fusion(SEQ ID NO: 23) between human tramdL sequences and humanSTK4/Mst-1/Krs-2 sequences (located on human chromosome 5 and humanchromosome 20 respectively). In some embodiments, the fusion is detectedby amplification of fusion cDNA sequences. The method comprises (a)providing (i) human parathyroid tumor tissue and (ii) tramdL andSTK4/Mst-1/Krs-2 primers; (b) isolating RNA from said parathyroid tumortissue; (c) reverse-transcribing said RNA to generate cDNA sequences;(d) using said primers to amplify said cDNA, and; (e) detecting anamplification product. In other embodiments, the fusion is detected byfluorescence in situ hybridization (FISH). The method comprises; (a)providing: (i) isolated tissue derived from human parathyroid tumors and(ii) differentially labeled tramdL and STK4/Mst-1/Krs-2 probes; (b)hybridizing said probes to said tissue, and; (c) detecting thehybridization pattern of said differentially labeled probes. In someembodiments, the method provides cells cultured from parathyroid tumorsin step (a). In some embodiments the tramdL probe comprises labeledsequences from a tramdL-containing bacterial artificial chromosome(BAC). In some embodiments, the label comprises biotin, while in otherembodiments the label comprises a fluorescent moiety, including but notlimited to fluorescein. In some embodiments, the STK4/Mst-1/Krs-2 probecomprises labeled sequences from a STK4/Mst-1/Krs-2 containing BAC. Insome embodiments, the label comprises digoxigenin, while in otherembodiments the label comprises a fluorescent moiety, including but notlimited to rhodamine.

[0019] In some embodiments, a transgenic animal model of parathyroidtumors, including parathyroid adenomas, is contemplated. In someembodiments, the transgenic animal is a transgenic mouse. The transgenicmouse comprises a recombinant construct wherein theSTK4/Mst-1/Krs-2-tramdL fusion is expressed under the control of asequence expressed primarily, if not exclusively, in the parathyroid.Thus, the transgenic animal comprises recombinant nucleic acidconstructs comprising the STK4/Mst-1/Krs-2-tramdL fusion (SEQ ID NO: 38)operably linked to a regulatory sequence which enableparathyroid-specific expression of the fusion. In some embodiments, theregulatory sequence is the Gcm2 promoter (the Gcm2 cDNA sequence has theaccession number AF081556 (SEQ ID NO: 24)), while in other embodiments,the regulatory sequence is the regulatory region of the mouseparathyroid hormone gene (mouse parathyroid hormone cDNA has theaccession number NM_(—)020623 (SEQ ID NO: 25) and mouse parathyroidhormone precursor, exons 2 and 3 cDNA has the accession number AF066075(SEQ ID NO: 33). In some embodiments, the Gcm2 coding region is replacedwith the STK4/Mst-1/Krs-2-tramdL fusion (SEQ ID NO: 38). In otherembodiments, the parathyroid hormone coding region is replaced with theSTK4/Mst-1/Krs-2-tramdL fusion (SEQ ID NO: 38). In some embodiments, thetransgenic animals are contemplated for the testing of therapeutics.Methods of generating transgenic animal models for parathyroid adenomasare also contemplated.

[0020] In some embodiments, human tramdorin sequences are contemplatedfor use in detection of disease-associated mutations in an autosomalrecessive Charcot-Maric-Tooth Syndrome demyelinating neuropathy whichmaps to 5q31-33. Human tramdorin exons can be isolated and sequenced insubjects with the disease and in subjects without the disease toidentify mutations in the affected subjects, which are not present inthe unaffected subjects.

[0021] In some embodiments, tramdorin is contemplated for use in assaysof Schwann cell myelination. In some embodiments, tramdorin isoverexpressed in isolated Schwann cells, which are co cultured withneurons. The rate of myelination is measured. In some embodiments,tramdorin is overexpressed from a recombinant expression vectorcomprising tramdorin cDNA. In some embodiments, said cDNA is selectedfrom the group consisting of SEQ ID NO: 13 and SEQ ID NO: 3. In someembodiments, the overexpressed tramdorin protein is mouse tramdorin 1(SEQ ID NO: 14), while in other embodiments, the overexpressed tramdorinis human tramdorin 1 (SEQ ID NO: 19). In some embodiments, myelinationis detected by Sudan black staining, while in other embodiments,myelination is detected by measuring Protein 0 (P0) expression.

[0022] In some embodiments, mice deficient in tramdorin 1 expression arecontemplated. In some embodiments, tramdorin 1 sequences are deleted inthe tramdorin 1-deficient mice, while in other embodiments, a portion ofthe tramdorin sequence is replaced by a marker gene, the expression ofwhich is detectable. In some embodiments, said marker gene is the LacZgene, the product of which, β-galactosidase, is detectable. In someembodiments, the deletion of tramdorin 1 sequences is mediated by aconditional knock-out of tramdorin 1 sequences, although a variety ofmeans to accomplish tramdorin 1 deletion are also contemplated.

[0023] In some embodiments, portions of tramdorin nucleic acid sequencesare contemplated for use in assays to detect tramdorin expression. Insome embodiments, said nucleic acid sequences comprise portions ofnucleic acid sequences selected from the group consisting of mousetramdorin 1 cDNA (SEQ ID NO: 13), mouse tramdorin 2 cDNA (SEQ ID NO: 1),mouse tramdorin 3 cDNA (SEQ ID NO: 2), human tramdorin 1 cDNA (SEQ IDNO: 3), human tramdorin 2 cDNA (SEQ ID NO: 4), human tramdorin 3 cDNA(SEQ ID NO: 5) and rat tramdorin 1 cDNA (SEQ ID NO: 6). In yet otherembodiments, said portions comprise 300 to 600 base pair fragments thatcontain the extreme C-terminal and 3′ untranslated sequences. In someembodiments, tramdorin expression is detected in tissues. In someembodiments, the detection uses in situ hybridization to sections offixed tissue. In some embodiments, the tissue comprises human tissue,while in other embodiments the tissue comprises mouse tissue, and instill other embodiments, the tissue comprises rat tissues. Any tissue iscontemplated, including but not limited to brain tissue, peripheralnervous tissue and spinal cord tissue. Any method of tissue fixation isalso contemplated, including formalin fixation.

[0024] In some embodiments, human tramdorin sequences are contemplatedfor use in the treatment of neuropathic pain.

[0025] In some embodiments, human tramdorin sequences are contemplatedfor use as a marker for bone growth, remodelling and/or osteoclastdifferentiation. In other embodiments, human tramdorin sequences arecontemplated to regulate bone growth.

[0026] In some embodiments, human tramdorin sequences are contemplatedfor use as a marker for adipose tissue. In other embodiments, humantramdorin sequences are contemplated to regulate the proliferationand/or differentiation of adipocytes. In other embodiments humantramdorin sequences are contemplated to regulate the sequestration ofglycine into adipocytes.

BRIEF DESCRIPTION OF THE FIGURES

[0027]FIG. 1 shows RDA clone JBSN125; a clone that contains two distinctsequences, one of which is differentially expressed in wild-type andOct-6 (−/−) siatic nerve and is homologous to multiple EST cDNA's. FIG.1A shows Southern blots of PCR-amplified cDNA from Oct-6 wild-type (+/+)and mutant (−/−) sciatic nerve. FIG. 1 B diagrammatically shows cDNAfragments that correspond to RDA clone JBSN 125-10, which contains onlydifferentially expressed sequences that are contained in JBSN125.

[0028]FIG. 2 shows the sequences of mouse tramdorin 1 EST cDNA 1920302(SEQ ID NO: 2), a putative rat tramdorin 1 cDNA (SEQ ID NO: 6), and thecorresponding amino acid sequences (SEQ ID NO: 14 and SEQ ID NO: 22).Image Consortium numbers are used for EST cDNAs.

[0029]FIG. 3 shows the structure predicted for the protein encoded bymouse tramdorin 1 EST cDNA 1920302. FIG. 3A shows the putative aminoacid sequence (SEQ ID NO: 14) encoded by the putative full length ESTcDNA 1920302, with structure predictions. FIG. 3B shows a diagramdepicting the predicted topology of tramdorin 1.

[0030]FIG. 4 shows a Northern blot depicting the tissue-specificexpression of mouse tramdorin 1.

[0031]FIG. 5 shows Northern blots depicting the expression of tramdorin1 and other genes at various times following nerve injury. FIG. 5A showstramdorin 1 expression following transection injury. FIG. 5B shows Oct-6expression following transection injury. FIG. 5C shows P0 expressionfollowing transection injury. FIG. 5D shows NGFR expression followingtransection injury. FIG. 5E shows GADPH expression following transectioninjury. FIG. 5F shows tramdorin expression following crush injury. FIG.5G shows Oct-6 expression following crush injury. FIG. 5H shows P0expression following crush injury. FIG. 5I shows NGFR expressionfollowing crush injury. FIG. 5J shows GADPH expression following crushinjury.

[0032]FIG. 6 summarizes the mouse mapping results for tramdorin. FIG. 6Asummarizes the mapping data. FIG. 6B shows a partial mouse chromosome 11linkage map.

[0033]FIG. 7 shows the cluster of four human tramdorin genes defined byhuman tramdorin EST cDNAs.

[0034]FIG. 8 shows the organization of the human tramdorin loci. FIG. 8Adepicts the human tramdorin 1 locus. FIG. 8B depicts the human tramdorin2 locus. FIG. 8C depicts the human tramdorin 3 locus. FIG. 8D depictsEST cDNA 1388139, which contains sequences that correspond to bothchromosome 5q and to the STK4/Mst-1/Krs-2 locus on chromosome 20.

[0035]FIG. 9 depicts the human genomic sequence of the tramdorin generegion (SEQ ID NO: 26). Sequence between nucleotides 50001 and 300000 ofHomo Sapiens chromosome 5 working draft sequence segment NT_(—)006951.4is shown.

[0036]FIG. 10 depicts the putative cDNA sequence for human tramdorin 1(SEQ ID NO: 3) and the corresponding amino acid sequence (SEQ ID NO:19).

[0037]FIG. 11 depicts the putative cDNA sequence for human tramdorin 2(SEQ ID NO: 4) and the corresponding amino acid sequence (SEQ ID NO:20).

[0038]FIG. 12 depicts human STK4/Mst-1/Krs-2 and tramdL sequences. FIG.12A depicts genomic sequence from nucleotides 8189548 to 8196472 ofhuman chromosome 20 contig NT011382 (SEQ ID NO: 27 (nucleotide sequence)and SEQ ID NO: 28 (corresponding amino acid sequence)). FIG. 12B depictshuman chromosome 5 genomic sequence between nts 55336 and 56128 (SEQ IDNO: 29) on Celera scaffold sequence Gax2HTBL3TT27:2500000-3000000 and anopen reading frame (SEQ ID NO: 30). FIG. 12C depicts the fusion junctionbetween the 5′ splice site of STK4/Mst-1/Krs-2 exon 9 and a 3′ splicesite of tramdorin L (SEQ ID NO: 31 (nucleotide sequence) and SEQ ID NO:32 (amino acid sequence)). FIG. 12D depicts diagrams of the proteinsencoded by STK4/Mst-1/Krs-2 and EST cDNA 1833139.

[0039]FIG. 13 depicts Northern blots of the tissue-specific expressionof human tramdorin genes. FIG. 13A shows tramdorin 1 expression. FIG.13B shows tramd 2 expression. FIG. 13C shows tramd3 expression. FIG. 13Dshows GADPH expression.

[0040]FIG. 14 depicts the conservation between the putative translationproducts of human tramdorin 1 (SEQ ID NO: 19), human tramdorin 2 (SEQ IDNO: 20), and human tramdorin 3 (SEQ ID NO: 21).

[0041]FIG. 15 depicts the conservation between putative tramdorin 1proteins from human (SEQ ID NO: 19), rat (SEQ ID NO: 22), mouse (SEQ IDNO: 14), Drosophila melanogaster (SEQ ID NO: 34) and C. elegans (SEQ IDNO: 35).

[0042]FIG. 16 depicts the conservation between human tramd 1 (SEQ ID NO:19), human tramd 3 (SEQ ID NO: 21), and a rat (SEQ ID NO: 36), confirmedexperimentally, and C. elegans (SEQ ID NO: 37), based on homology, VGAT.

[0043]FIG. 17 depicts the organization of mouse tramdorin loci. Exonsare denoted as boxes; filled boxes denote coding regions. Coordinatesdenote the location of the sequences on Celera mouse genome scaffoldsequence GA_xK02T2QP88:1-500000. A. Mouse tramd1. A partial restrictionmap is shown at the top of the figure. Shown below the map are threetramd1 EST cDNAs that define tramd1 exons. Genomic regions that weresequenced prior to the availability of the Celera mouse genome sequenceare show as thick lines below the cDNAs, and below them, selectsubcloned fragments are shown. B. Mouse tramd2. Two mouse tramdorin2RACE cDNA clones are shown. C. Mouse tramd3. A partial restriction mapis shown at the top of the figure. Exons 2-8 were defined by homology tomouse tramd1 or human tramd3. Three mouse tramdorin3 cDNA clones areshown; two are RACE clones, and one was amplified with tramd3-specificprimers. Regions that were sequenced prior to the availability of theCelera mouse genome sequence are shown below the exons; these sequenceswere obtained from the ends of the cloned fragments shown at the bottomof the figure, or from the Ensemb1 mouse genome sequence database. Notethat tramdorin3 is transcribed in the opposite direction from tramdorin1and tramdorin2.

[0044]FIG. 18 depicts the genomic sequence of mouse tramdorin 1 (SEQ IDNO: 7).

[0045]FIG. 19 depicts the genomic sequence of mouse tramdorin 3 (SEQ IDNO: 8).

[0046]FIG. 20 depicts the conservation between human (SEQ ID NO: 21) andmouse tramd 3 (SEQ ID NO: 18) proteins.

[0047]FIG. 21 depicts the composite human tramd 3 cDNA sequence (SEQ IDNO: 5) and the corresponding amino acid sequence (SEQ ID NO: 21).

[0048]FIG. 22 depicts the composite mouse tramdorin 2 cDNA (SEQ IDNO: 1) sequence and the corresponding amino acid sequence (SEQ ID NO:17).

[0049]FIG. 23 depicts mouse tramd3 cDNA sequences. FIG. 23A depicts acomposite mouse tramd 3 cDNA (SEQ ID NO: 2). FIG. 23B depicts mousetramd 3 3′ RACE cDNA 58#37 (SEQ ID NO: 39). FIG. 23C depicts mouse tramd3 3′ RACE cDNA 56#22 (SEQ ID NO: 40).

[0050]FIG. 24 depicts the rat LYAAT-1 mRNA,and its translation. FIG. 24Adepicts the translation of rat LYAAT-1, Genebank Accesion No.AAK67316.1, (SEQ ID NO: 41). FIG. 24B depicts the rat LYAAT-1, GenebankAccesion No. AF361239, mRNA (SEQ ID NO: 42).

[0051]FIG. 25 depicts the tramdL EST cDNA 1388139 sequence (SEQ ID NO:23) and the corresponding amino acid sequence (SEQ ID NO: 71).

[0052]FIG. 26 depicts the predicted cDNA sequence of aSTK4/Mst-1/Krs-2-tramdL fusion containing 5′ STK4 sequences not includedin EST cDNA 1388139 (SEQ ID NO: 38) and the corresponding amino acidsequence (SEQ ID NO: 44).

[0053]FIG. 27 depicts mouse Gcm2 and Pth cDNA sequences. FIG. 27A showsthe mouse Gcm2 cDNA sequence, Accession AF081556 (SEQ ID NO: 24). FIG.27B shows mouse parathyroid hormone cDNA, Accession NM_(—)02063 (SEQ IDNO: 25). FIG. 27C depicts mouse parathyroid hormone precursor, exons 2,3 and cDNA AF066075 (SEQ ID NO: 33).

[0054]FIG. 28 depicts the phylogenetic relationship of tramd1 to othercloned genes. Specifically, this dendrogram illustrates that tramd1 isclosely related to, but distinct from tramd3/LYAAT-1, and that theseproteins along with the hypothetical proteins CG13384 from Drosophilamelanogaster, and T27A1.5 from Caenorhabditis elegans, constitutes a newfamily of putative amino acid transport proteins. They are related tothe yeast vacuolar amino acid transport proteins AVT3 and AVT4. OtherAVT proteins, and vesicular GABA transport proteins are more distantlyrelated. The glycine transporters Glyt1 and Glyt2 are shown also forcomparison.

[0055]FIG. 29 depicts immunoblot analysis of tramdorin1. Specificallythis figure presents a Western blot analysis of anti-tramdorin1antiserum. 100 μg of lysates from (see, panel A) adult rat sciatic nerveand (see, panel B) 293 cells transfected with tramdorin1 expressionvector (+), and untransfected 293 cells (−) were used for western blotanalysis of the tramdorin1 antiserum (diluted 1:1000); the blots weredeveloped with chemiluminescence. See, panels C and D. Micrographsshowing transfected (panel C) or untransfected (panel D) 293 cellsviewed by immunofluorescence using the anti-tramdorin1 antiserum. Nucleiwere visualized using DAPI.

[0056]FIG. 30 depicts localization of tramdorin1 in myelinating Schwanncells. These are images of unfixed teased fibers from adult rat sciaticnerve, double-labeled with a rabbit tramdorin1 antiserum (see, panels Aand D; TRITC) and a mouse monoclonal antibody against MAG (see, panel B;FITC) or LAMP1 (see, panel E; FITC). Tramdorin-immunoreactivity is foundat paranodes (arrows) and incisures (arrowheads), colocalizing with MAG(panels A-C), and in puncta along the outer surface. At paranodes(panels D-F), tramdorin1-immunostaining does not co-localize with thatof LAMP1, a lysosomal marker.

[0057]FIG. 31 depicts darkfield micrographs of mouse bone and fat padtissue hybridized with probes specific for tramd1, tramd2, and tramd3.

[0058]FIG. 32 depicts the nucleic acid sequence of the mouse tramd1probe (SEQ ID NO: 71) used in the in situ hybridizations presented inFIG. 31.

[0059]FIG. 33 depicts the nucleic acid sequence of the mouse tramd2probe (SEQ ID NO: 72) used in the in situ hybridizations presented inFIG. 31.

[0060]FIG. 34 depicts the nucleic acid sequence of the mouse tramd3probe (SEQ ID NO: 73) used in the in situ hybridizations presented inFIG. 31.

DEFINITIONS

[0061] As used herein, “tramdorin”, “tramd” or “tramdorins” and “tramds”refers to a family of related genes and related proteins that have beenidentified in at least human, mouse and rat. Tramdorin genes encodeputative transmembrane proteins. At least four tramdorin family membersexist: tramdorin 1 (tramd1), tramdorin 2 (tramd2), tramdorin 3 (tramd3)and tramdL.

[0062] Tramdorin 1 has been identified in human, mouse and rat. The cDNAsequences for each are presented in FIG. 10 (putative cDNA for humantramdorin1 (SEQ ID NO: 3)), FIG. 2 (mouse tramdorin1 cDNA (SEQ ID NO:13)) and FIG. 2 (putative rat tramdorin 1 cDNA (SEQ ID NO: 6)). Themouse tramdorin 1 genomic sequence is presented in FIG. 18 (SEQ ID NO:7). The genomic sequence for human tramdorin 1 (SEQ ID NO: 9) iscontained within the sequence presented in FIG. 9 (SEQ ID NO: 26).Specifically, the human tramdorin 1 gene is predicted to be containedwithin nucleotides 214333-256951 of FIG. 9, although it is alsocontemplated that additional regulatory sequences may lie outside ofthis span of nucleotides.

[0063] Tramd2 has been identified in human and mouse. The genomicsequence of human tramd 2 (SEQ ID NO: 10) is contained within thesequence presented in FIG. 9 (SEQ ID NO: 26). Specifically, the humantramdorin 2 gene is predicted to be contained within nucleotides258170-294569 of FIG. 9, although it is also contemplated thatadditional regulatory regions may lie outside of this span ofnucleotides. The cDNA sequence encoding tramd2 in human is presented inFIG. 11 (putative human tramd2 cDNA sequence (SEQ ID NO: 4)) and thecDNA sequence encoding tramd 2 in mouse is presented in FIG. 22(composite mouse tramdorin2 cDNA (SEQ ID NO: 1)).

[0064] Tramdorin3 has also been identified in human, rat and mouse. Thegenomic sequence encoding tramd3 in human (SEQ ID NO: 11) is containedin the sequence presented in FIG. 9 (SEQ ID NO: 26) (note that tramd3 isshown in the antisense orientation in this figure). Specifically, thehuman tramdorin 3 gene is predicted to be contained within nucleotides58749-136740 of FIG. 9, although it is also contemplated that additionalregulatory regions may lie outside of this span of nucleotides. Thegenomic sequence encoding tramd 3 in mouse is shown in FIG. 19 (genomicsequence of mouse tramdorin 3 (SEQ ID NO: 8)). The cDNA sequenceencoding human tramd 3 is presented in FIG. 21 (composite human tramd 3cDNA (SEQ ID NO: 5)). The coding region of the cDNA sequence encodingmouse tramd 3 is shown in FIG. 23A (composite mouse tramd 3 cDNA (SEQ IDNO: 2)). The mRNA sequence (SEQ ID NO: 42), and its translation (SEQ IDNO: 41), of rat LYAAT-1 (isolated by Sagne et al., PNAS USA 98:7206(2001)), which appears to be a rat homolog of tramd 3 is presented inFIG. 24.

[0065] An additional tramdorin family member has been identified inhuman, tramdL, which exists as a splice junction fusion between atramdorin 3′ exon (SEQ ID NO: 29) (FIG. 12B) and 5′ STK4/Mst-1/Krs-2sequences encoded on human chromosome 20 (SEQ ID NO: 27) (FIG. 12A). ThecDNA sequence of the fusion junction is shown in FIG. 12C (SEQ ID NO:31), and the putative STK4/Mst-1/Krs-2-tramdL fusion protein is depictedin FIG. 12D. The cDNA sequence of EST cDNA 1388139 (SEQ ID NO: 23) isdepicted in FIG. 25, as is the corresponding predicted amino acidsequence (SEQ ID NO: 71).

[0066] With respect to nucleic acid sequences, one of skill in the artwill appreciate that poplymorphisms are often found in populations, andany such polymorphisms contained within any of the tramdorin genomic orcDNA sequences are also within the scope of the present invention.

[0067] The predicted proteins encoded by human tramdorins 1 (SEQ ID NO:19), 2 (SEQ ID NO: 20) and 3 (SEQ ID NO: 21) are depicted in FIG. 14.The putative amino acid sequence encoded by mouse tramd1 cDNA isdepicted in FIG. 3 (SEQ ID NO: 14). The amino acid sequences for human(SEQ ID NO: 21) and mouse (SEQ ID NO: 18) tramd3 proteins are shown inFIG. 20. In addition to the human, rat and mouse tramdorins describedabove, tramdorins appear to exist in other species. For example,homologous sequences have been identified in Drosophila melanogaster(FIG. 15) and Caenorhabditis elegans (FIG. 15). Thus one of skill in theart will understand that tramdorin family members may be identifiedbased on homology to the sequences described above.

[0068] The putative amino acid sequence (corresponding to the sequencelabeled “1920303” in FIG. 3A) encoded by mouse tramd1 cDNA is depictedin FIG. 3 (SEQ ID NO: 14). This sequence has been assigned GenBankaccession number AF12429. That is to say, the amino acid sequencereferenced by GenBank accession number AF12429 is identical to thesequence labeled “1920303” in FIG. 3A. This mouse cDNA encodes a 478AAprotein.

[0069] The amino acid sequences for human (SEQ ID NO: 21) and mouse (SEQID NO: 18) tramd3 proteins are shown in FIG. 20. In addition to thehuman, rat and mouse tramdorins described above, tramdorins appear toexist in other species. For example, homologous sequences have beenidentified in Drosophila melanogaster (FIG. 15) and Caenorhabditiselegans (FIG. 15). Thus one of skill in the art will understand thattramdorin family members may be identified based on homology to thesequences described above.

[0070] As used herein, “derivatives” of tramdorin proteins can refer toa number of alterations in such proteins. In some embodiments, thederivatives comprise proteins with amino acid sequence changes. Suchchanges can be conservative amino acid substitutions, amino aciddeletions or amino acid insertions, provided that the activity of thetramdorin is retained. Preferably, the alterations are conservativeamino acid changes. For example, it is contemplated that an isolatedreplacement of a leucine with an isoleucine or valine, an alanine with aglycine, a threonine with a serine or a similar replacement of an aminoacid with a structurally related amino acid (i.e. conservativesubstitutions) will not have a major effect on the biological activityof the resulting molecule. Conservative substitutions are those thattake place within a family of amino acids that are related by their sidechains. Amino acids can be divided into four families: (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3)nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In an alternative, yet similar fashion, the amino acidrepertoire can be grouped as: (1) acidic (aspartate, glutamate); (2)basic (lysine, arginine, histidine); (3) aliphatic (glycine, alanine,valine, leucine, isoleucine, serine, threonine), with serine andthreonine optionally grouped separately as aliphatic-hydroxyl; (4)aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine,glutamine); and (6) sulfur-containing (cysteine and methionine) (Seee.g. Stryer ed., Biochemistry, 2E, W H Freeman and Co. (1981) pp.13-16). Thus, in certain embodiments, modifications of the tramdorinprotein sequence(s) is contemplated by the present invention. Guidancein determining which and how many amino acid residues may besubstituted, inserted, or deleted without abolishing biological activitymay be found by using computer programs well known in the art, forexample, DNAStar software or GCG (Univ. of Wisconsin).

[0071] As used herein, “central nervous system”, or “CNS” refers to thebrain and spinal cord. The tissues of the CNS comprise both neurons andaccessory, or “glial” cells. The accessory cells of the CNS are referredto as “oligodendrocytes”, which form insulating myelin sheaths aroundthe axons of many neurons, and “astrocytes” which provide structural andmetabolic support for neurons and also induce tight junctions betweencells lining the capillaries in the brain.

[0072] As used herein, the “peripheral nervous system”, or “PNS” refersto the nerves and supporting cells that communicate motor and sensorysignals between the CNS and the rest of the body. The supporting cellsof the PNS are referred to as “Schwann cells”, which form the insulatingmyelin sheaths around the axons of many neurons.

[0073] As used herein, “myelin” refers to the lipoproteinaceousmaterial, composed of regularly alternating membranes of lipid lamellae(consisting of cholesterol, phospholipids, sphingolipids,phosphatidates) and protein, of the myelin sheath.

[0074] As used herein, the term “demyclinating disease” refers to anypathological process that results in the degradation or loss of themyelin sheath surrounding an axon, including, but not limited toMultiple Sclerosis, Charcot-Marie Tooth (CMT) and Guillain-Barresyndrome.

[0075] As used herein, the term “gene” refers to a DNA sequence thatcomprises control and coding sequences necessary for the production of apolypeptide or protein precursor. The polypeptide can be encoded by afull length coding sequence or by any portion of the coding sequence, aslong as the desired protein activity is retained. “Nucleoside”, as usedherein, refers to a compound consisting of a purine [guanine (G) oradenine (A)] or pyrimidine [thymine (T), uridine (U), or cytidine (C)]base covalently linked to a pentose, whereas “nucleotide” refers to anucleoside phosphorylated at one of its pentose hydroxyl groups.

[0076] A “nucleic acid”, as used herein, is a covalently linked sequenceof nucleotides in which the 3′ position of the pentose of one nucleotideis joined by a phosphodiester group to the 5′ position of the pentose ofthe next, and in which the nucleotide residues (bases) are linked inspecific sequence; i.e., a linear order of nucleotides. A“polynucleotide”, as used herein, is a nucleic acid containing asequence that is greater than about 100 nucleotides in length. An“oligonucleotide”, as used herein, is a short polynucleotide or aportion of a polynucleotide. An oligonucleotide typically contains asequence of about two to about one hundred bases. The word “oligo” issometimes used in place of the word “oligonucleotide”. Nucleic acidmolecules are said to have a “5′-terminus” (5′ end) and a “3′-terminus”(3′ end) because nucleic acid phosphodiester linkages occur to the 5′carbon and 3′ carbon of the pentose ring of the substituentmononucleotides. The end of a nucleic acid at which a new linkage wouldbe to a 5′ pentose carbon is its 5′ terminal nucleotide (by conventionsequences are written, from right to left, in the 5′ to 3′ direction).The end of a nucleic acid at which a new linkage would be to a 3′pentose carbon is its 3′ terminal nucleotide. A terminal nucleotide, asused herein, is the nucleotide at the end position of the 3′- or5′-terminus. DNA molecules are said to have “5′ ends” and “3′ ends”because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. Therefore, an end of an oligonucleotide isreferred to as the “5′ end” if its 5′ phosphate is not linked to the 3′oxygen of a mononucleotide pentose ring and as the “3′ end” if its 3′oxygen is not linked to a 5′ phosphate of a subsequent mononucleotidepentose ring.

[0077] As used herein, a nucleic acid sequence, even if internal to alarger oligonucleotide or polynucleotide, also may be said to have 5′and 3′ ends. In either a linear or circular DNA molecule, discreteelements are referred to as being “upstream” or 5′ of the “downstream”or 3′ elements. This terminology reflects the fact that transcriptionproceeds in a 5′ to 3′ fashion along the DNA strand. Typically, promoterand enhancer elements that direct transcription of a linked gene aregenerally located 5′ or upstream of the coding region. However, enhancerelements can exert their effect even when located 3′ of the promoterelement and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

[0078] The term “wild-type” when made in reference to a gene refers to agene which has the characteristics of a gene isolated from a naturallyoccurring source. The term “wild-type” when made in reference to a geneproduct refers to a gene product which has the characteristics of a geneproduct isolated from a naturally occurring source. A wild-type gene isthat which is most frequently observed in a population and is thusarbitrarily designated the “normal” or “wild-type” form of the gene. Incontrast, the term “modified” or “mutant” when made in reference to agene or to a gene product refers, respectively, to a gene or to a geneproduct which displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct. For some variants (i.e. modified or mutant forms of a gene),there is no selection against individuals carrying the variant form, andso both forms (wild-type and modified) may be relatively common in thegeneral population. This is referred to as “genetic polymorphism”, whichis defined herein as a genetic variation in which the frequency of twoor more forms, or alleles, is at least 1% in the population.Polymorphisms within the tramdorin sequences are specificallycontemplated.

[0079] As used herein in reference to a nucleic acid sequence (forexample a cDNA or genomic sequence), the term “portion” (as in “aportion of a genomic sequence”) refers to fragments of that nucleic acidsequence. The fragments may range in size from five nucleotides to theentire nucleic acid sequence minus one nucleotide.

[0080] The term “antisense” as used herein refers to adeoxyribonucleotide sequence whose sequence of deoxyribonucleotideresidues is in reverse 5′ to 3′ orientation in relation to the sequenceof deoxyribonucleotide residues in a sense strand of a DNA duplex.Antisense also refers to the “reverse complement orientation”. By way ofexample, the reverse complement of the nucleotide sequence 5′-GATCC-3′is 5′-CTAGG-3′. A “sense strand” of a DNA duplex refers to a strand in aDNA duplex which is transcribed by a cell in its natural state into a“sense mRNA.” Thus an “antisense” sequence is a sequence having the samesequence as the non-coding strand in a DNA duplex. The term “antisenseRNA” refers to a RNA transcript that is complementary to all or part ofa target primary transcript or mRNA and that blocks the expression of atarget gene by interfering with the processing, transport and/ortranslation of its primary transcript or mRNA. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. In addition, as used herein, antisense RNA maycontain regions of ribozyme sequences that increase the efficacy ofantisense RNA to block gene expression. “Ribozyme” refers to a catalyticRNA and includes sequence-specific endoribonucleases. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of preventing the expression of the target protein.

[0081] As used herein, the term “overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. As used herein,the term “altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

[0082] The term “recombinant” when made in reference to a DNA moleculerefers to a DNA molecule which is comprised of segments of DNA joinedtogether by means of molecular biological techniques. The term“recombinant” when made in reference to a protein or a polypeptiderefers to a protein molecule which is expressed using a recombinant DNAmolecule.

[0083] The term “nucleotide sequence of interest” refers to anynucleotide sequence, the manipulation of which may be deemed desirablefor any reason (e.g., confer improved qualities), by one of ordinaryskill in the art. Such nucleotide sequences include, but are not limitedto, coding sequences of structural genes (e.g., reporter genes,selection marker genes, oncogenes, drug resistance genes, growthfactors, etc.), and non-coding regulatory sequences which do not encodean mRNA or protein product, (e.g., promoter sequence, polyadenylationsequence, termination sequence, enhancer sequence, etc.).

[0084] As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. Typically, the coding region is bounded on the 5′side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by a stop codon (e.g., TAA, TAG, TGA). Insome cases the coding region is also known to initiate by a nucleotidetriplet “TTG”.

[0085] As used herein, the terms “complementary” or “complementarity”when used in reference to polynucleotides refer to polynucleotides whichare related by the base-pairing rules. For example, for the sequence5′-AGT-3′ is complementary to the sequence 5′-ACT-3′. Complementaritymay be “partial,” in which only some of the nucleic acids′ bases arematched according to the base pairing rules. Or, there may be “complete”or “total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods which depend upon binding between nucleicacids.

[0086] A “complement” of a nucleic acid sequence as used herein refersto a nucleotide sequence whose nucleic acids show total complementarityto the nucleic acids of the nucleic acid sequence.

[0087] The term “homology” when used in relation to nucleic acids refersto a degree of complementarity. There may be partial homology orcomplete homology (i. e., identity). “Sequence identity” refers to ameasure of relatedness between two or more nucleic acids or proteins,and is given as a percentage with reference to the total comparisonlength. The identity calculation takes into account those nucleotide oramino acid residues that are identical and in the same relativepositions in their respective larger sequences. Calculations of identitymay be performed by algorithms contained within computer programs suchas “GAP” (Genetics Computer Group, Madison, Wis.) and “ALIGN” (DNAStar,Madison, Wis.). A partially complementary sequence is one that at leastpartially inhibits (or competes with) a completely complementarysequence from hybridizing to a target nucleic acid is referred to usingthe functional term “substantially homologous.” The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (Southern orNorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or probe willcompete for and inhibit the binding (i.e., the hybridization) of asequence which is completely homologous to a target under conditions oflow stringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target which lacks even a partialdegree of complementarity (e.g., less than about 30% identity); in theabsence of non-specific binding the probe will not hybridize to thesecond non-complementary target.

[0088] When used in reference to a double-stranded nucleic acid sequencesuch as a cDNA or genomic clone, the term “substantially homologous”refers to any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described infra.

[0089] Low stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,5×Denhardt's reagent [50×Denhardt's contains per 500 ml: 5 g Ficoll(Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42° C. when a probe of about 500 nucleotides inlength is employed.

[0090] High stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

[0091] When used in reference to nucleic acid hybridization the artknows well that numerous equivalent conditions may be employed tocomprise either low or high stringency conditions; factors such as thelength and nature (DNA, RNA, base composition) of the probe and natureof the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution maybe varied to generate conditions of either low or high stringencyhybridization different from, but equivalent to, the above listedconditions.

[0092] “Stringency” when used in reference to nucleic acid hybridizationtypically occurs in a range from about T_(m)−5° C. (5° C. below theT_(m) of the probe) to about 20° C. to 25° C. below T_(m). As will beunderstood by those of skill in the art, a stringent hybridization canbe used to identify or detect identical polynucleotide sequences or toidentify or detect similar or related polynucleotide sequences. Under“stringent conditions” a nucleic acid sequence of interest willhybridize to its exact complement and closely related sequences.

[0093] Polypeptide molecules are said to have an “amino terminus”(N-terminus) and a “carboxy terminus” (C-terminus) because peptidelinkages occur between the backbone amino group of a first amino acidresidue and the backbone carboxyl group of a second amino acid residue.Typically, the terminus of a polypeptide at which a new linkage would beto the carboxy-terminus of the growing polypeptide chain, andpolypeptide sequences are written from left to right beginning at theamino terminus.

[0094] As used herein in reference to an amino acid sequence or aprotein, the term “portion” (as in “a portion of an amino acidsequence”) refers to fragments of that protein. The fragments may rangein size from four amino acid residues to the entire amino acid sequenceminus one amino acid.

[0095] As used herein, the term “fusion protein” refers to a chimericprotein containing the protein of interest (e.g., tramdorins andfragments thereof) joined to an exogenous protein fragment (e.g., thefusion partner which consists of a non-tramdorin protein). The fusionpartner may enhance the solubility of tramdorin protein as expressed ina host cell, may provide an affinity tag to allow purification of therecombinant fusion protein from the host cell or culture supernatant, orboth. The fusion partner may also enable screening assays, such as theyeast two-hybrid assay. If desired, the fusion protein may be removedfrom the protein of interest (e.g., tramdorins or fragments thereof) bya variety of enzymatic or chemical means known to the art.

[0096] The term “isolated” when used in relation to a nucleic acid, asin “an isolated nucleic acid sequence” refers to a nucleic acid sequencethat is identified and separated from at least one contaminant nucleicacid with which it is ordinarily associated in its natural source.Isolated nucleic acid is nucleic acid present in a form or setting thatis different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA whichare found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs which encode a multitude of proteins. However,an isolated nucleic acid sequence encoding human tramdorin 1 (forexample) includes, by way of example, such nucleic acid sequences incells which ordinarily contain the sequence encoding human tramdorin 1,where the nucleic acid sequence is in a chromosomal or extrachromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid sequence may be present in single-stranded ordouble-stranded form. When an isolated nucleic acid sequence is to beutilized to express a protein, the nucleic acid sequence will contain ata minimum at least a portion of the sense or coding strand (i.e., thenucleic acid sequence may be single-stranded). Alternatively, it maycontain both the sense and anti-sense strands (i.e., the nucleic acidsequence may be double-stranded).

[0097] As used herein, the term “purified” refers to molecules, eithernucleic or amino acid sequences, that are removed from their naturalenvironment, isolated or separated. An “isolated nucleic acid sequence”is therefore a purified nucleic acid sequence. “Substantially purified”molecules are at least 60% free, preferably at least 75% free, and morepreferably at least 90% free from other components with which they arenaturally associated.

[0098] As used herein, the terms “vector” and “vehicle” are usedinterchangeably in reference to nucleic acid molecules that transfer DNAsegment(s) from one cell to another. Vectors may include plasmids,bacteriophages, viruses, cosmids, and the like.

[0099] The term “expression vector” or “expression cassette” as usedherein refers to a recombinant DNA molecule containing a desired codingsequence and appropriate nucleic acid sequences necessary for theexpression of the operably linked coding sequence in a particular hostorganism. Nucleic acid sequences necessary for expression in prokaryotesusually include a promoter, an operator (optional), and a ribosomebinding site, often along with other sequences. Eukaryotic cells areknown to utilize promoters, enhancers, and termination andpolyadenylation signals.

[0100] The terms “targeting vector” and “targeting construct” refer tonucleic acid sequences comprising a sequence of interest flanked oneither side by recognition sequences that are capable of homologousrecombination with cognate sequences in the genome in such a way thatthe sequence of interest replaces any DNA sequences that are locatedbetween the cognate sequences in the genome. The sequence of interestcan consist of recognition sites for restriction enzymes or sitespecific recombinases such as Flp or Cre, exongenous genes including butnot limited to those that encode thymidinekinase or confer resistance toantibiotics such as neomycin and hygromycin, marker genes such as LacZand eGFP, as well as portions of the targeted gene itself or sequencesfrom other genes of interest.

[0101] The terms “in operable combination”, “in operable order” and“operably linked” as used herein refer to the linkage of nucleic acidsequences in such a manner that a nucleic acid molecule capable ofdirecting the transcription of a given gene and/or the synthesis of adesired protein molecule is produced. The term also refers to thelinkage of amino acid sequences in such a manner so that a functionalprotein is produced.

[0102] The term “selectable marker” as used herein, refer to a genewhich encodes an enzyme having an activity that confers resistance to anantibiotic or drug upon the cell in which the selectable marker isexpressed. Selectable markers may be “positive” or “negative.” Examplesof positive selectable markers include the neomycin phosphotrasferase(NPTII) gene which confers resistance to G418 and to kanamycin, and thebacterial hygromycin phosphotransferase gene (hyg), which confersresistance to the antibiotic hygromycin. Negative selectable markersencode an enzymatic activity whose expression is cytotoxic to the cellwhen grown in an appropriate selective medium. For example, the HSV-tkgene is commonly used as a negative selectable marker. Expression of theHSV-tk gene in cells grown in the presence of gancyclovir or acycloviris cytotoxic; thus, growth of cells in selective medium containinggancyclovir or acyclovir selects against cells capable of expressing afunctional HSV TK enzyme.

[0103] Transcriptional control signals in eukaryotes comprise “promoter”and “enhancer” elements. Promoters and enhancers consist of short arraysof DNA sequences that interact specifically with cellular proteinsinvolved in transcription (Maniatis, et al., Science 236:1237, 1987).Promoter and enhancer elements have been isolated from a variety ofeukaryotic sources including genes in yeast, insect, mammalian and plantcells. Promoter and enhancer elements have also been isolated fromviruses and analogous control elements, such as promoters, are alsofound in prokaryotes. The selection of a particular promoter andenhancer depends on the cell type used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types (forreview, see Voss, et al., Trends Biochem. Sci., 11:287, 1986; andManiatis, et al., supra 1987).

[0104] The terms “promoter element,” “promoter,” or “promoter sequence”as used herein, refer to a DNA sequence that is located at the 5′ end(i.e. precedes) the protein coding region of a DNA polymer. The locationof most promoters known in nature precedes the transcribed region. Thepromoter functions as a switch, activating the expression of a gene. Ifthe gene is activated, it is said to be transcribed, or participating intranscription. Transcription involves the synthesis of mRNA from thegene. The promoter, therefore, serves as a transcriptional regulatoryelement and also provides a site for initiation of transcription of thegene into mRNA.

[0105] Promoters may be tissue specific or cell specific. The term“tissue specific” as it applies to a promoter refers to a promoter thatis capable of directing selective expression of a nucleotide sequence ofinterest to a specific type of tissue in the relative absence ofexpression of the same nucleotide sequence of interest in a differenttype of tissue. Tissue specificity of a promoter may be evaluated by,for example, operably linking a reporter gene to the promoter sequenceto generate a reporter construct, introducing the reporter constructinto the genome of an organism such that the reporter construct isintegrated into every tissue of the resulting transgenic organism, anddetecting the expression of the reporter gene (e.g., detecting mRNA,protein, or the activity of a protein encoded by the reporter gene) indifferent tissues of the transgenic. The detection of a greater level ofexpression of the reporter gene in one or more tissues relative to thelevel of expression of the reporter gene in other tissues shows that thepromoter is specific for the tissues in which greater levels ofexpression are detected. The term “cell type specific” as applied to apromoter refers to a promoter which is capable of directing selectiveexpression of a nucleotide sequence of interest in a specific type ofcell in the relative absence of expression of the same nucleotidesequence of interest in a different type of cell within the same tissue.The term “cell type specific” when applied to a promoter also means apromoter capable of promoting selective expression of a nucleotidesequence of interest in a region within a single tissue. Cell typespecificity of a promoter may be assessed using methods well known inthe art, e.g., immunohistochemical staining. Briefly, tissue sectionsare embedded in paraffin, and paraffin sections are reacted with aprimary antibody which is specific for the polypeptide product encodedby the nucleotide sequence of interest whose expression is controlled bythe promoter. A labeled (e.g., peroxidase conjugated) secondary antibodywhich is specific for the primary antibody is allowed to bind to thesectioned tissue and specific binding detected (e.g., withavidin/biotin) by microscopy. Alternatively, mRNA localization can bedetermined by in situ hybridization.

[0106] Promoters may be constitutive or regulatable. The term“constitutive” when made in reference to a promoter means that thepromoter is capable of directing transcription of an operably linkednucleic acid sequence in the absence of a stimulus (e.g., heat shock,chemicals, light, etc.). Typically, constitutive promoters are capableof directing expression of a transgene in substantially any cell and anytissue.

[0107] In contrast, a “regulatable” promoter is one which is capable ofdirecting a level of transcription of an operably linked nuclei acidsequence in the presence of a stimulus (e.g., heat shock, chemicals,light, etc.) which is different from the level of transcription of theoperably linked nucleic acid sequence in the absence of the stimulus.

[0108] As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequence(s). For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc.

[0109] The enhancer and/or promoter may be “endogenous” or “exogenous”or “heterologous.” An “endogenous” enhancer or promoter is one that isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” enhancer or promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of the gene isdirected by the linked enhancer or promoter. For example, an endogenouspromoter in operable combination with a first gene can be isolated,removed and placed in operable combination with a second gene, therebymaking it a “heterologous” promoter in operable combination with saidsecond gene. A variety of such combinations are contemplated (e.g. thefirst and second genes can be from the same species, or from differentspecies).

[0110] The presence of “splicing signals” on an expression vector oftenresults in higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site (Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, New York [1989] pp.16.7-16.8). A commonly used splice donor and acceptor site is the splicejunction from the 16S RNA of SV40.

[0111] Efficient expression of recombinant DNA sequences in eukaryoticcells requires expression of signals directing the efficient terminationand polyadenylation of the resulting transcript. Transcriptiontermination signals are generally found downstream of thepolyadenylation signal and are a few hundred nucleotides in length. Theterm “poly(A) site” or “poly(A) sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable, as transcripts lacking a poly(A) tail areunstable and are rapidly degraded. The poly(A) signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly(A) signal is one that is found naturally at the 3′ end of thecoding region of a given gene in the genome. A heterologous poly(A)signal is one which has been isolated from one gene and positioned 3′ toanother gene. A commonly used heterologous poly(A) signal is the SV40poly(A) signal. The SV40 poly(A) signal is contained on a 237 bpBamHI/BclI restriction fragment and directs both termination andpolyadenylation (Sambrook, supra, at 16.6-16.7).

[0112] The term “transfection” as used herein refers to the introductionof foreign DNA into eukaryotic cells. Transfection may be accomplishedby a variety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.When introducing foreign DNA into yeast cells, the term “transformation”is commonly used.

[0113] The term “transgenic” when used in reference to a cell refers toa cell which contains a transgene, or whose genome has been altered bythe introduction of a transgene. The term “transgenic” when used inreference to a tissue or to an organism, such as a mouse, refers to atissue or organism, respectively, which comprises one or more cells thatcontain a transgene, or whose genome has been altered by theintroduction of a transgene. Transgenic cells, tissues and organisms maybe produced by several methods including the introduction of a“transgene” comprising nucleic acid (usually DNA) into a target cell orintegration of the transgene into a chromosome of a target cell by wayof human intervention, such as by the methods described herein.

[0114] The term “transgene” as used herein refers to any nucleic acidsequence which is introduced into the genome of a cell by experimentalmanipulations. A transgene may be an “endogenous DNA sequence,” or a“heterologous DNA sequence” (i.e., “foreign DNA”). The term “endogenousDNA sequence” refers to a nucleotide sequence which is naturally foundin the cell into which it is introduced so long as it does not containsome modification (e.g., a point mutation, the presence of a selectablemarker gene, etc.) relative to the naturally-occurring sequence. Theterm “heterologous DNA sequence” refers to a nucleotide sequence whichis ligated to, or is manipulated to become ligated to, a nucleic acidsequence to which it is not ligated in nature, or to which it is ligatedat a different location in nature. Heterologous DNA is not endogenous tothe cell into which it is introduced, but has been obtained from anothercell. Heterologous DNA also includes an endogenous DNA sequence whichcontains some modification. Generally, although not necessarily,heterologous DNA encodes RNA and proteins that are not normally producedby the cell into which it is expressed. Examples of heterologous DNAinclude reporter genes, transcriptional and translational regulatorysequences, selectable marker proteins (e.g., proteins which confer drugresistance), etc.

[0115] The term “foreign gene” refers to any nucleic acid (e.g., genesequence) which is introduced into the genome of a cell by experimentalmanipulations and may include gene sequences found in that cell so longas the introduced gene contains some modification (e.g., a pointmutation, the presence of a selectable marker gene, etc.) relative tothe naturally-occurring gene.

[0116] Transformation of a cell may be stable or transient. The term“transient transformation” or “transiently transformed” refers to theintroduction of one or more transgenes into a cell in the absence ofintegration of the transgene into the host cell's genome. Transienttransformation may be detected by, for example, enzyme-linkedimmunosorbent assay (ELISA) which detects the presence of a polypeptideencoded by one or more of the transgenes. Alternatively, transienttransformation may be detected by detecting the activity of the protein(e.g., β-glucuronidase) encoded by the transgene. The term “transienttransformant” refers to a cell which has transiently incorporated one ormore transgenes. In contrast, the term “stable transformation” or“stably transformed” refers to the introduction and integration of oneor more transgenes into the genome of a cell. Stable transformation of acell may be detected by Southern blot hybridization of genomic DNA ofthe cell with nucleic acid sequences which are capable of binding to oneor more of the transgenes. Alternatively, stable transformation of acell may also be detected by the polymerase chain reaction of genomicDNA of the cell to amplify transgene sequences. The term “stabletransformant” refers to a cell which has stably integrated one or moretransgenes into the genomic DNA. Thus, a stable transformant isdistinguished from a transient transformant in that, whereas genomic DNAfrom the stable transformant contains one or more transgenes, genomicDNA from the transient transformant does not contain a transgene.

[0117] The term “amplification” is defined as the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction technologies well known in the art(Dieffenbach and G S Dvekler, PCR Primer, a Laboratory Manual, ColdSpring Harbor Press, Plainview N.Y. [1995]). As used herein, the term“polymerase chain reaction” (“PCR”) refers to the methods disclosed inU.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, all of which areincorporated herein by reference, which describe a method for increasingthe concentration of a segment of a target sequence in a mixture ofgenomic DNA without cloning or purification. This process for amplifyingthe target sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed totheir complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one “cycle”; there canbe numerous “cycles”) to obtain a high concentration of an amplifiedsegment of the desired target sequence. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of the primers with respect to each other, and therefore, thislength is a controllable parameter. By virtue of the repeating aspect ofthe process, the method is referred to as the “polymerase chainreaction” (hereinafter “PCR”). Because the desired amplified segments ofthe target sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified.”

[0118] With PCR, it is possible to amplify a single copy of a specifictarget sequence in genomic DNA to a level detectable by severaldifferent methodologies (e.g., hybridization with a labeled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; and/or incorporation of ³²P-labeleddeoxyribonucleotide triphosphates, such as dCTP or dATP, into theamplified segment). hi addition to genomic DNA, any oligonucleotidesequence can be amplified with the appropriate set of primer molecules.In particular, the amplified segments created by the PCR process itselfare, themselves, efficient templates for subsequent PCR amplifications.Amplified target sequences may be used to obtain segments of DNA (e.g.,genes) for the construction of targeting vectors, transgenes, etc.

[0119] As used herein, the term “sample template” refers to a nucleicacid originating from a sample which is analyzed for the presence of“target”. In contrast, “background template” is used in reference tonucleic acid other than sample template, which may or may not be presentin a sample. Background template is most often inadvertent. It may bethe result of carryover, or it may be due to the presence of nucleicacid contaminants sought to be purified away from the sample. Forexample, nucleic acids other than those to be detected may be present asbackground in a test sample.

[0120] As used herein, the term “primer” refers to an oligonucleotide,whether occurring naturally (e.g., as in a purified restriction digest)or produced synthetically, which is capable of acting as a point ofinitiation of nucleic acid synthesis when placed under conditions inwhich synthesis of a primer extension product which is complementary toa nucleic acid strand is induced (i.e., in the presence of nucleotides,an inducing agent such as DNA polymerase, and under suitable conditionsof temperature and pH). The primer is preferably single-stranded formaximum efficiency in amplification, but may alternatively bedouble-stranded. If double-stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and useof the method.

[0121] As used herein, the term “probe” refers to a polynucleotidesequence (for example an oligonucleotide), whether occurring naturally(e.g., as in a purified restriction digest) or produced synthetically,recombinantly or by PCR amplification, which is capable of hybridizingto another nucleic acid sequence of interest. A probe may besingle-stranded or double-stranded. Probes are useful in the detection,identification and isolation of particular gene sequences. It iscontemplated that the probe used in the present invention is labeledwith any “reporter molecule,” so that it is detectable in a detectionsystem, including, but not limited to enzyme (i.e., ELISA, as well asenzyme-based histochemical assays), fluorescent, radioactive, andluminescent systems. It is not intended that the present invention belimited to any particular detection system or label. The terms “reportermolecule” and “label” are used herein interchangeably. In addition toprobes, primers and deoxynucleoside triphosphates may contain labels;these labels may comprise, but are not limited to, ³²P, ³³P, 35S,enzymes, or fluorescent molecules (e.g., fluorescent dyes).

[0122] As used herein, “FISH” refers to fluorescence in situhybridization, a technique in which detectably labeled DNA probes (whichcan be prepared, for example, from cDNA sequences or genomic sequencescontained in cosmids or bacterial artificial chromosomes (BACs)) arehybridized to cytogenetic or histological specimens. Such specimensinclude, but are not limited to, metaphase chromosome spreads andinterphase nuclei prepared from tissue or blood specimens, andformaldehyde-fixed, paraffin-embedded tissue sections. Fluorescentlabels can be directly incorporated into the probe, or can be applied asantibody-label conjugates which bind to affinity labels (for example,biotin or digoxigenin) incorporated into the probe, either directly, oras an antibody “sandwich” (i.e. a primary and a secondary antibody). Thefluorescent dyes include, but are not limited to rhodamine, texas red,FITC (fluorescein isothiocyanate) and TRITC (tetramethyl rhodamineisothiocyanate). The fluorescent labels are detected using afluorescence microscope equipped with a mercury or xenon lamp (as anillumination source) and appropriate filters for excitation andemission. The pattern of fluorescence can be used to assess copy numberof the locus recognized by the probe, or, in cases where two or more(differentially) labeled probes are used, to assess the relativepositions of the probes (for example to detect chromosomalrearrangements, such as translocations and inversions).

[0123] As used herein, “parathyroid tumors” refers to tumors (benign ormalignant) of the parathyroid gland. By way of example, a parathyroidadenoma is a benign tumor of the parathyroid gland. Other parathyroidtumors include parathyroid carcinomas.

[0124] As used herein, “parathyroid adenoma” refers to a benign tumor ofa parathyroid gland. This enlargment and overactivity of the affectedgland results in continued production of parathyroid hormone(hyperparathyroidism). In turn, elevated levels of calcium are found inthe blood.

[0125] As used herein, the term “regulatory region of a gene expressedin the parathyroid” refers to the upstream regulatory sequences(including but not limited to promoter sequences) of a gene that isexpressed primarily (if not exclusively) in the parathyroid gland. Suchgenes (and their regulatory regions) include, but are not limited to theparathyroid hormone gene and the Gcm2 gene. Such regulatory regions,when placed in operable combination with a sequence of interest willpermit expression of the sequence of interest in a tissue-specificfashion in the parathyroid.

[0126] As used herein, “POU domain transcription factor” refers to amember of a family of transcription factors, characterized by possessionof a bipartite DNA binding domain (the POU domain) that also providesbinding sites for many proteins that interact with POU domains. POUdomain transcription factors include, but are not limited to thetranscription factor Oct-6 (also known as Tst-1 or SCIP).

[0127] As used herein, a “STK4/Mst-1/Krs-2-tramd L fusion” or“STK4/Mst-1/Krs-2-tramdorin L fusion” refers to a fusion betweentramdorin L sequences on human chromosome 5 and STK4/Mst-1/Krs-2sequences on human chromosome 20. The fusion may be at the level ofgenomic sequences, or at the level of cDNA sequences, or both. Thefusion involves at least the known exon of tramdL, as shown in FIG. 12B,and at least the genomic DNA encoding exons 6-9 of STK4/Mst-1/Krs-2, asshown in FIG. 12A. STK4/Mst-1/Krs-2 is a member of the STE20 family ofkinases. The STK4/Mst-1/Krs-2-tramd L fusion, as represented in EST cDNA1388139 (SEQ ID NO: 23), comprises the STK4/Mst-1/Krs-2 kinase domainsequences fused to 3′ sequences of tramd L. A sequence of arepresentative STK4/Mst-1/Krs-2-tramdL fusion junction (in a cDNA) isshown in FIG. 12C (SEQ ID NO: 31). The genomic sequences from theSTK4/Mst-1/Krs-2 and tramdL partners of a representative fusion areshown in FIGS. 12A (SEQ ID NO: 27) and 12B (SEQ ID NO: 29),respectively. A predicted cDNA (SEQ ID NO: 38) and protein sequence (SEQID NO: 44) of a fusion containing additional 5′ STK4 sequences notcontained in EST cDNA 1388139 is provided in FIG. 26.

[0128] As used herein, “NGFR” refers to a nerve growth factor receptorgene.

[0129] As used herein, “GADPH” refers to a glyceraldehydes-3-phosphatedehydrogenase gene.

[0130] As used herein, “Sos” (an acronym for “son-of-sevenless”) refersto a ras guanine nucleotide exchange factor.

[0131] As used herein, “GABA” refers to y-aminobutyric acid.

[0132] As used herein, “Gcm2” refers to the glial cells missing proteinhomolog (Gcm2) gene.

[0133] As used herein, the term “syntenic” refers to the preservation of“blocks” of genes in some fashion across species. That is, syntenicrefers to a region of conserved synteny with another species, wherein“synteny” refers to the physical presence together on the samechromosome of two or more gene loci, whether or not they are closeenough together for linkage to be demonstrated.

[0134] As used herein, the term “incisures” (also referred to as“Schmidt-Lanterman incisures”), are regions of non-compact myelin (i.e.regions of the myelin sheath that contain cytoplasm).

[0135] As used herein, the term “paranode” refers to a region adjacentto the node of Ranvier (i.e. the junction between adjacent myelinsheathes). The paranode contains a set of proteins that are not found inthe node itself, or in the internode, the myelinated region betweennodes. A single Schwann cell will contain half a node of Ranvier, aparanode, an internode, another paranode, and another half a node ofRanvier.

[0136] As used herein, “KLH” refers to the protein called Keyhole LimpetHemocyanin.

DETAILED DESCRIPTION OF THE INVENTION

[0137] Mouse tramdorin 1 was identified in a screen for target genes ofthe POU domain transcription factor, Oct-6. Tramd1 encodes a putativetransmembrane domain protein, and the expression pattern of tramd1suggests that it is related to myelination in the peripheral nervoussystem. Homology searching identified tramdorin 1 homologues in humanand rat, as well as identified a family of closely-related tramdoringenes, with at least four members. The location of the tramdorin genecluster in humans is suggestive of a potential role in a peripheralneuropathy. One of the family members is present as a gene fragment, andmay play a role in the etiology of the parathyroid tumors which is, inone embodiment, an adenoma. Compositions comprising the tramdorins(nucleic acid and amino acid sequences) and host cells transfected withtramdorins are contemplated. Additionally, the use of tramdorins in avariety of diagnostic assays is contemplated, as is the development of atransgenic mouse model for parathyroid tumors. The description of theinvention includes I. The initial identification and characterization ofmouse tramdorin 1, II. Identification of tramdorin homologs in mouse andother species, III. Organization of the tramdorin family locus in mouseand human, IV. Functional characterization of tramdorins V. Tramdorinsand parathyroid tumors and VI. Tramdorins and peripheral neuropathies.VII. Remyelination by Schwann cells in the CNS following disease orinjury.

I. The Initial Characterization of Mouse Tramdorin 1

[0138] Tramdorin 1 was identified in experiments directed to the studyof myclination in the peripheral nervous system. Myelin sheathes aremembranous structures that surround large caliber axons. By limitingaxonal depolarization to the nodes of Ranvier that separate individualsheaths, myelin permits rapid axonal conductance over long distanceswith small axonal volumes, a property that is essential for normalfunctioning of vertebrate nervous systems. The debilitating effects ofdiseases that affect myelin, such as Charcot Marie Tooth Syndrome andmultiple sclerosis, demonstrate its importance. Myelin is produced bySchwann cells in the PNS and by oligodendrocytes in the CNS. Both celltypes ensheathe axons, then wrap them with multiple layers of membranethat, following exclusion of cytoplasm and compaction, constitute themyelin sheathes. However, the molecular mechanisms that control thesynthesis of myelin are poorly understood.

[0139] Schwann cells are the primary glial cells of the peripheralnervous system. Myelinating Schwann cells form a single myclin sheatharound a single axon, whereas non-myelinating Schwann cells typicallyensheathe several unmyelinated axons. Axons regulate the proliferationof Schwann cells, and in addition, they are thought to regulate theirdifferentiation: presumptive myelinated axons signal Schwann cells intothe myelinating lineage, whereas the axons lacking this signal remainassociated with non-myelinating Schwann cells. Intracellular cAMPsignaling is thought to play a central role in axonal control of bothSchwann cell proliferation and differentiation, although the nature ofthe signal(s) that induce elevation of the levels of cyclic AMP (cAMP)in Schwann cells remain unknown. In cultured Schwann cells, many aspectsof axon-induced Schwann cell proliferation and differentiation can bemimicked by activators of adenylyl cyclase, such as forskolin. Forskolintreatment decreases expression of genes that are associated withnon-myelinating Schwann cells and immature Schwann cells, and increasesexpression of genes associated with myelinating Schwann cells, includingthe POU domain transcription factor Oct-6 and the zinc-fingertranscription factor Krox-20. These data indicate that cAMP may be asecond messenger of the axonal signal that promotes myelination, andthat the change to the myelinating phenotype is mediated by changes inthe expression of several transcription factors. Both Oct-6 and Krox-20play essential roles in the development of myelinating Schwann cells[Bermingham et al., Gene Develop 10:1751 (1996)]. However, the preciserole played by cAMP signaling in Schwann cell myelination is not yetfully understood.

[0140] Oct-6 (also known as Tst-1 or SCIP) is a member of the POU domainfamily of transcription factors. These proteins are characterized bytheir possession of a bipartite DNA binding domain (the POU domain) thatalso provides binding sites for many proteins that interact with POUproteins. Oct-6 is expressed transiently during Schwann cell development[Monuki et al., Neuron 3:783 (1989)], specifically in promyelinatingSchwann cells, which have formed a 1:1 relation with axons but have notyet formed a myelin sheath. Oct-6 represses expression of severalmyelin-related promoters in transient cotransfection assays, and on thisbasis, has been hypothesized to repress myclination. It is alsoexpressed transiently in oligodendrocytes [Collarini et al., Development116:193 (1992)]. The phenotypes of mutations in Oct-6 indicate that itcan control the timing of myelination. In the absence of Oct-6, Schwanncell differentiation is delayed, transiently arrested at thepromyelinating stage [Bermingham et al., Gene Develop 10:1751 (1996) andJaego, et al., Science 273:507 (1996)]. CNS myelination appears normal,perhaps due to the presence in oligodendrocytes, but not Schwann cells,of two other members of the class III POU domain family, Brn-1 and Brn-2[Schreiber et al., J Biol Chem 272:32286 (1997)]. Transgenic mice thatexpress a truncated form of Oct-6 in Schwann cells display precociousperipheral myelination [Weinstein et al., Mol Cell Neurosci 6:212(1995)], and Oct-6 misexpression in oligodendrocytes causes precociousCNS myelination and hypomyelination [Jensen et al., J Clin Invest101:1292 (1998)]. Oct-6 has been hypothesized to activate genes requiredfor Schwann cell differentiation, then to repress terminaldifferentiation.

[0141] The identification of target genes that are regulated by Oct-6would enhance our understanding of its role in Schwann cell development.Recently, representational difference analysis (RDA; [Lisitsyn, TrensGenet 11:303 (1995)], a PCR-based technique that permits the isolationof DNA fragments that are present in one DNA sample but absent inanother, was used to identify genes that are differentially expressed insciatic nerve from Oct-6 homozygous mutant (−/−) and wild-type (+/+)mice. Six cDNA fragments were identified that correspond to genes thatare activated by Oct-6; no repressed genes were identified, suggestingthat Oct-6 functions as an activator in Schwann cells [Bermingham etal., manuscript in preparation]. Of these genes, only one has beendescribed previously in Schwann cells, and three are novel. Theidentities of two known putative target genes suggest cytoskeletalrearrangement or fatty acid transport as rate-limiting steps inmyelination that are activated by Oct-6. One of the previously unknowngenes named Tramdorin 1 (transmembrane domain rich protein). Tramdorin 1has been assigned the human and mouse gene symbol tramd1.

[0142] RDA clone JBSN125 contained tramdorin1 sequences that weredifferentially expressed between Oct-6 (−/−) and wild-type (+/+) sciaticnerve, as well as a sequence whose expression was unaffected by theabsence of Oct-6. A subclone of this, 125-10, contained onlydifferentially expressed sequences and was used to isolate additionalsequence for the tramdorin1 gene.

[0143] Mouse cDNAs that correspond to clone 125-10 were isolated byobtaining and sequencing three homologous EST clones. Mouse EST cDNA1920302 defines a 2.5 kb cDNA (SEQ ID NO: 13/GenBank Acession No.: AI1780664). One of the EST clones appears to correspond to a partiallyspliced transcript. The full-length mouse cDNA contains a 1.4 kb openreading frame (FIG. 2) that encodes a protein of 52 kd. The mouse cDNAand the putative protein that it encodes do not match any genes in theGenBank non-redundant database, or the SwissProt database. Therefore,these cDNAs are derived from a novel gene. That is to say, there are noexact matches to the protein sequence.

[0144] The protein encoded by mouse cDNA 1920302 (SEQ ID NO: 13) waspredicted by several protein structure prediction programs to encode atransmembrane protein (see examples below). Regardless of the differingpredictions about the number of transmembrane domains, or theirtopology, this protein appears to contain multiple transmembranehelices.

[0145] To determine the size of tramdorin mRNA(s) and to determine iftramdorin expression is restricted to sciatic nerve, a northern blotwith RNAs from adult mouse sciatic nerve and other tissues washybridized to a radiolabelled probe synthesized from tramdorin cDNA1920302 (SEQ ID NO: 13). Tramdorin1 expression is neither ubiquitous,nor restricted to sciatic nerve (see examples below).

[0146] It was of interest to determine whether the expression of tramd1in sciatic nerve is modulated by nerve injury. A northern blot of RNAfrom sciatic nerves collected at various times following crush ortransection injury was hybridized with a mouse tramd1 probe (seeexamples below). Tramd1 expression is downregulated by transectioninjury, and remains low. Following a crush injury, tramd1 isdownregulated, but subsequently it is reexpressed as axons regenerateand are remyelinated. Therefore, the tramdorin1 gene encodes amyelin-related protein. Several genes that are required for normalmyelination show this pattern of expression; these include Oct-6, P0,connexin32 and PMP-22 [Podsulo, Regulation of myelin gene expression inthe peripheral nervous system. In Dyck, P. J. et al. (eds.), PeripheralNeuropathy, W. B. Saunders Co., Philadelphia, [1993] pp. 282-289;Scherer et al., J Neurosci 14:1930 (1994); Sohl et al., Eur J Cell biol69:267 (1996)]. This result suggests that tramd1 may play an importantrole in the timely formation of peripheral myelin. Thus, an importantfunction of Oct-6 in myelination may be activating the expression oftramdorin 1, a gene that encodes a novel transmembrane domain protein.Additionally, the expression of tramd1 following nerve injury indicatesthat it will be useful as a marker of myelinating Schwann cells.Following crush injuries, Schwann cells distal to the injurydownregulate expression of myelin-related genes such as P0, and expressmarkers of immature Schwann cells, such as p75 nerve growth factor[Poduslo, “Regulation of myelin gene expression in the peripheralnervous system.” In Dyck, P. J. et al. (eds), Peripheral Neuropathy, W.B. Saunders Co., Philadelphia, pp. 282-289 (1993)]. As axons regenerate,they are remyelinated, with concomitant re-expression of myclin-relatedgenes. Tramdorin1 shows this pattern of expression. Therefore thepresent invention contemplates using tramdorin 1 to augment currentmarkers of myelinating Schwann cells.

[0147] Remyelination of axons in the CNS by Schwann cells has beenexplored extensively as a possible treatment for demyelinating diseasessuch as multiple sclerosis, and as a treatment for nerve injury. MSaffects 1/1000 Americans, and in people of northern European descent,the frequency is 1/800 [Lynch et al., Dis Mon 42:1 (1996)]; itrepresents a significant public health problem in the United States andEurope. An understanding of the genes that regulate myelination bySchwann cells is needed for optimizing their use in remyelination in theCNS. While it is not necessary to understand the underlying mechanismsto practice the invention (and the invention is not limited to anyparticular mechanism), it is believed that the tramdorin 1 gene may playa crucial role in regulating myelination for the following reasons:First, it is a putative target gene for Oct-6, a transcription factorthat is essential for timely peripheral myelination [Bermingham et al.,Gene Develop, 10:1751 (1996); Jaegle et al., Science 273:507 (1996)].Second, its expression following nerve injury indicates that it encodesa myelin-related protein. Screens using tramdorin, and/or its putativeligands, will provide avenues for the development of therapeutic agentsfor the treatment of the effects of demyelination or nerve injury, bothin the peripheral and central nervous systems. The ability to identifymolecules that promote myelination will significantly enhance theprospects for successfully using Schwann cells, and perhapsoligodendrocytes and olfactory ensheathing cells as well, to mitigatethe damage caused by MS and other demyclinating diseases. A significantuse of tramdorin 1 will be in identifying such molecules (see sectionIV, “Functional Characterization of Tramdorins”).

[0148] The mouse chromosomal location of Tramd1 was determined byinterspecific backcross analysis using progeny derived from matings of[(C57BL/6J x Mus spretus)F₁ X C57BL/6J] mice (see examples below). Themapping results indicated that Tramd1 is located in the proximal regionof mouse chromosome 11 linked to Il13 and Hand1. The most likely geneorder is: centromere-Il13-1/142-Tramd1-6/179-Hand1. The recombinationfrequencies (expressed as genetic distances in centiMorgans (cM)±thestandard error) are-Il13-0.7+/−0.7-Tramd1-3.4+/−1.4-Hand1. The proximalregion of mouse chromosome 11 is syntenic with human chromosome 5q31-33.An autosomal recessive Charcot-Marie-Tooth Syndrome demyelinatingneuropathy has been mapped to this region [Guilbot et al., Ann NY AcadSci 883:56 (1999a); Guilbot et al., Eur J Hum Genet 7:849 (1999b);LeGuern et al., Hum Mol Genet 5:1685 (1996)], suggesting that tramdorin1could be a candidate peripheral neuropathy disease gene.

II. Identification of Tramdorin Homologs in Rat and Other Species

[0149] A. Rat

[0150] A Rat cDNA that corresponds to mouse clone 125-10 was isolated by5′ and 3′ RACE (rapid isolation of cDNA ends; [Frohman, Methods Enzymol218:340 (1993); Frohman, PCR Methods Appl 4: S40 (1994)]). The 5′ and 3′rat RACE fragments combined define a 2.5 kb (SEQ ID NO: 55) cDNA. Thefull-length rat cDNA (SEQ ID NO: 6) contains a 1.4 kb open reading frame(FIG. 2) that encodes a protein of 52 kd (SEQ ID NO: 22). The rat cDNAand the putative protein that it encodes (FIGS. 2 and 15) does not matchany genes in the GenBank non-redundant database, or the SwissProtdatabase. Therefore, this cDNA is derived from a novel gene.

[0151] A Northern blot using rat tissue did not show hybridization tolung and thymus (data not shown), suggesting that there may be variationin tramdorin 1 expression among species, or cross-hybridization torelated sequences in the mouse Northern blots.

[0152] B. Human

[0153] As noted above, the proximal region of mouse chromosome 11 issyntenic with human chromosome 5q31-33, suggesting that the humanhomolog of tramd1 will map to 5q31-33 as well. An autosomal recessiveCharcot-Marie-Tooth Syndrome demyelinating neuropathy has been mapped tothis region, suggesting that tramdorin 1 could be a candidate peripheralneuropathy disease gene.

[0154] As a first step toward determining if tramdorin1 mutations areassociated with human peripheral neuropathy, the human tramdorin genewas studied. Six human EST cDNAs that possess homology to mousetramdorin1, I.M.A.G.E numbers 3184556 (BE501426), 1738130 (AI140615),1837427 (AI208756), 1388139 (AA843982), and 2549054 (AI953890), as wellas EST cDNA DKFZp434G1123 from the Deutsches Ressourcezentrum fürGenomforschung GmbH (RZPD) were obtained and sequenced in theirentirety. The sequences were aligned on human genomic DNA from theInternational Human Genome Sequencing Consortium (IHGSC) and Celeradatabases. These cDNAs showed greatest homology to four discretelocations within 250 kb of chromosome 5q sequence (FIG. 7), confirmingthe mapping of tramdorin in mouse, but in addition, suggesting theexistence of multiple tramdorin genes (see examples below). These geneshave been assigned the names tramd1 (SEQ ID NO: 9), tramd2 (SEQ ID NO:10), tramd3 (SEQ ID NO: 11), and tramdL (SEQ ID NO: 12). The predictedcDNA and encoded amino acid sequences of human tramd1 (cDNA: SEQ ID NO:3; corresponding amino acid sequence: SEQ ID NO: 19), tramd2 (cDNA: SEQID NO: 4; corresponding amino acid sequence: SEQ ID NO: 20) and tramd3(cDNA: SEQ ID NO: 5; corresponding amino acid sequence: SEQ ID NO: 21)are shown in FIGS. 10, 11, and 21, respectively. The amino acid sequenceof human tramd3 (SEQ ID NO: 21) is also depicted in FIG. 20 (alignedwith mouse tramd3), and the amino acid sequences of human tramd 1 (SEQID NO: 19), tramd 2 (SEQ ID NO: 20) and tramd 3 (SEQ ID NO: 21) are alsodepicted in FIGS. 14, 15, 16 (tramd1 ), FIG. 14 (tramd 2) and FIGS. 14,16, 20 (tramd 3).

[0155] A fourth tramdorin-related sequence, tramdL, is defined by theEST cDNA 1388139 (SEQ ID NO: 23) (FIGS. 8D and 12). tramdL contains onlysequences that are homologous to exon 10 of tramdorin1. This cDNA isderived from a parathyroid adenoma, and is unusual in that its 5′ endconsists of sequences derived from a member of the STE20 family ofkinases, STK4/Mst-1/Krs-2 (FIG. 12A; [Creasy et al., J Biol Chem270:21695 (1995); Taylor et al., PNAS USA 93:10099 (1996)]), located onhuman chromosome 20. The STK4/Mst-1/Krs-2 cDNA fragment is fused to 3′sequences of tramdL (FIG. 12B), located on chromosome 5. Thisobservation raises the possibility that tramdL resides near aparathyroid tumor translocation breakpoint, between chromosomes 20 and5, that results in expression of a STK4/Mst-1/Krs-2-tramdorinL fusionprotein. Such a fusion protein may be relevant in the etiology ofparathyroid tumors, such as parathyroid adenomas, and severalapplications involving the use of this fusion in the evaluation ofparathyroid tumors, including but not limited to parathyroid adenomas,and in the development of an animal model of parathyroid tumors,including but not limited to parathyroid adenomas, are contemplated (seeSection V, Tramdorins and Parathyroid Tumors and Examples below).

[0156] To determine the tissues in which the various tramdorin genes areexpressed in humans, a northern blot of RNA derived from 12 humantissues was sequentially hybridized to radiolabelled cDNA probes thatcorrespond to human tramd1, 2, 3 and tramdL that were constructed asfollows:

[0157] htramd1 probe: Probe consists of 355 bp EcoRI-PvuII fragment ofEST cDNA 3184556. The Eco RI site resides in the vector that containsthe cDNA; the probe contains human tramd1 exons 7 and 8, and part of theintron between exons 8 and 9, ending at the PvuII site at position248018 in FIG. 9.

[0158] htramd2 probe: Probe consists of 440 bp EcoRI-NcoI fragment ofEST cDNA 1837427. The Eco RI site resides in the vector that containsthe cDNA; the probe contains sequences from position 1209 to the Ncosite at position 1636 in FIG. 11, and contains part of human tramd2 exon9, exon 10, and part of exon 11.

[0159] htramd3 probe: Probe consists of 850 bp StuI-KpnI fragment of ESTcDNA 2549054, consisting of the sequences between the Stul site atposition 232 and the Kpn site at position 1080 on FIG. 21.

[0160] htramdL probe: Probe consists of 340 bp MscI-DraI fragment of ESTcDNA 1388139, consisting of the sequences between the MscI site 7 bp 3′to the 3′ splice site of tramdL and the DraI site at the 5′ end of thepolyA tail of this cDNA, which is marked by an asterisk in FIG. 12B.

[0161] Each probe produced a distinct hybridization result (see examplebelow). Translation of the known putative coding exons for humantramdorins 1, 2, and 3 indicate that these proteins are highly conserved(see example below).

[0162] C. Tramdorins in Other Organisms

[0163] The most closely related sequences to tramdorins are putativeproteins that have been derived from the Drosophila, Caenorhabditiselegans and Saccaromyces cerevisiae genome sequences (see examplebelow). Mouse tramd1 protein is 39% identical to Drosophila proteinCG13384, and 32% identical to the T27A1.5 protein from C. elegans. Thefunctions of these proteins in invertebrates may be related to tramdorinfunction in vertebrates. If so, tramdorins would be required for afunction that is conserved by evolution.

[0164] Seven novel yeast amino acid transporter genes, Avt1-7, wereidentified by virtue of their homology to vesicular GABA transporter(VGAT) sequences (Russnak et al., J Biol Chem 276:23849 (2001)).VGAT/VIAAT/Unc-47 proteins are the known proteins that are most relatedto tramdorin/LYAAT proteins (FIG. 16). A CLUSTAL W comparison of humanand mouse tramd1, human tramd2, human tramd3 and AVT1-4 and AVT7proteins demonstrates that the tramdorins are most closely related toAVT3 and AVT4 (data not shown). Most of the AVT proteins, including AVT3and AVT4, were localized to vacuoles, a yeast organelle that closelyresembles mammalian lysosomes, consistent with the localization oftramd3/LYAAT1 to lysosomes (Sagne et al, PNAS USA 98:7206 (2001)). AVT3,4, and 6 appear to be involved in amino acid export from vacuoles,suggesting that tramdorins could perform a similar function inlysosomes. However, the observation that not all AVT proteins appear tobe localized exclusively in vacuoles suggests that other mammalianhomologs of these proteins may exist in different subcellular locations.

[0165] D. Structure of Tramd Proteins

[0166] While an understanding of the precise structure of tramdorins isnot necessary to the practice of the present invention, a number ofconsiderations suggest that tramd proteins consist of 11 transmembranedomains, with the N-terminus facing the cytoplasm (see example below).The vertebrate proteins to which the tramdorins are most closely relatedare vesicular γ-aminobutyric acid (GABA) transporters (VGATs; seeexample below; [McIntire et al., Nature 389:870 (1997); Sagne et al.,FEBS Lett 417:177 (1997)]).

III. Organization of the Tramdorin Locus in Human and Mouse

[0167] A. Human

[0168] As noted above, human EST cDNAs that possess homology to mousetramdorin 1 were aligned on human genomic DNA from the InternationalHuman Genome Sequencing Consortium (IHGSC) Homo sapiens chromosome 5working draft sequence segment NT_(—)006951.4 and Celera databases[Lander et al., Nature 409:860 (2001); Venter et al., Science 291:1304(2001)]. These cDNAs showed greatest homology to four discrete locationswithin 250 kb of chromosome 5q sequence (FIG. 7), confirming the mappingof tramdorin in mouse, but in addition, suggesting the existence ofmultiple tramdorin genes. These genes have been assigned the namestramd1, tramd2, tramd3, and tramdL. The human tramd1 (SEQ ID NO: 9) genehas greatest homology to the original mouse tramd1 (SEQ ID NO: 7) gene,and is flanked on its 3′ end by tramd2 (SEQ ID NO: 10), and on its 5′end by tramd3 (SEQ ID NO: 11), which is transcribed in the oppositeorientation to tramd1 and tramd2. Nested between tramd1 and tramd3resides a tramdorin gene fragment, tramdL (SEQ ID NO: 12), in the sameorientation as tramd1 and tramd2. It consists only of 3′ tramdorinsequences, and is defined by a single EST cDNA, 1388139 (SEQ ID NO: 23).All four putative genes are found on both the Celera and IHGSCsequences. FIG. 8 presents the organization of the human tramdorin loci(see Example below for details of the organization). The cDNAs in FIG. 8are aligned with Celera human chromosome 5 scaffold sequence GAx2HTBL3TT27:2500000-3000000, except where noted in the figure. Thegenomic sequence of 250 kb of sequence from the entire tramdorin region(nucleotides 50001-300000 of the IHGSC Homo Sapiens chromosome 5 workingdraft sequence segment NT_(—)006951.4) is presented in FIG. 9. Knowntramdorin exons are shown in bold type, and indicated to the right ofthe sequence. Initiation ATG and stop codons, and putativepolyadenylation signal sites are shown in bold italic. Note that tramd3is in the opposite orientation relative to the other tramd genes, and isshown in the antisense orientation.

[0169] Details of the structure of the individual tramdorin genes arepresented in the example below. Briefly, the structure of humantramdorin 1 (SEQ ID NO: 9) was determined by identifying regions ofhomology between mouse and human tramdorins, and by cDNA sequences. FIG.10 depicts the putative human tramd 1 cDNA (SEQ ID NO: 3) and encodedprotein (SEQ ID NO: 19). Human tramdorin 2 (SEQ ID NO: 4) exons aredefined by a single EST cDNA and by homology with tramdorins 1 (SEQ IDNO: 3) and 3 (SEQ ID NO: 5), and mouse tramd2 (SEQ ID NO: 1) cDNA. FIG.11 depicts the putative cDNA (SEQ ID NO: 3) and encoded protein (SEQ IDNO: 20) for human tramd 2. The sequences of four alternatively splicedtramdorin 3 EST cDNAs were used to infer the structure of the tramdorin3 gene. FIG. 21 depicts a composite human tramd3 cDNA (SEQ ID NO: 5) andcorresponding amino acid sequence (SEQ ID NO: 21). A fourthtramdorin-related sequence, tramdL (SEQ ID NO: 12), is defined by an ESTcDNA which is derived from a parathyroid adenoma. TramdL containssequences which are homologous to exon 10 of tramdorin 1. The 5′ end ofthis EST cDNA consists of sequences derived from a member of the STE20family of kinases, STK4/Mst-1/Krs-2 [Creasy et al., J Biol Chem270:21695 (1995); Taylor et al., PNAS USA 93:10099 (1996)], located onchromosome 20. The STK4/Mst-1/Krs-2 cDNA fragment is fused to 3′sequences of tramdL, located on chromosome 5. This observation raisesthe possibility that tramdL resides near a parathyroid tumortranslocation breakpoint, between chromosomes 20 and 5, that results inexpression of a STK4/Mst-1/Krs-2-tramdorinL fusion protein (see SectionV, Tramdorins and Parathyroid Tumors below).

[0170] B. Mouse

[0171] The organization of the mouse tramd1 gene (SEQ ID NO: 7) wasstudied to understand better the origin of the alternatively splicedtramdorin cDNAs, and as a preliminary step toward generating mice thatlack tramd1 function. A 129 mouse genomic library in bacteriophage λ wasscreened with a probe consisting of a 720 bp XhoI-NcoI fragment fromcDNA 1920302 (SEQ ID NO: 13) that contains sequence from exons 1-6.Three phage were isolated (FIG. 17A). In addition, a bacterialartificial chromosome (BAC) library (Research Genetics) was screenedusing the polymerase chain reaction (PCR) using primers that amplify 169bp of exon 6 (see example below). A single tramdorin-containing BAC wasisolated. Subclones from the BAC and from the lambda phage weresequenced to determine the structure of the 5′ end of the tramdoringene. The genomic sequence 3′ to exon 7 corresponds to sequence in ESTcDNA 1363993 (SEQ ID NO: 43), indicating that this cDNA is partiallyspliced. Exons 8, 9, and 10 are defined by sequences from the Ensemb1database. Mouse tramdorin 1 genomic sequences (SEQ ID NO: 7) arepresented in FIG. 18.

[0172] To determine if the BAC contained genomic sequences foradditional mouse tramdorin genes, plasmids that contained shotgun-clonedBAC DNA were arrayed on dot blots. Sequence analysis of clones thathybridized to radiolabelled tramd1 cDNA probes revealed that in additionto tramd1 sequences, the BAC also contained tramdorin 3 sequences (FIG.17B). Mouse tramdorin 3 genomic sequences (SEQ ID NO: 8) are presentedin FIG. 19, and a comparison between mouse (SEQ ID NO: 18) and humantramdorin 3 (SEQ ID NO: 21) proteins is presented in FIG. 20. BecauseBACs typically contain approximately 100 kb of DNA, the presence oftramd 1 and tramd3 sequences in the same BAC suggests that these genesmay reside more closely to one another in mice than they do in humans.

[0173] Composite mouse tramd3 cDNA (SEQ ID NO: 3) is presented in FIG.23. As described in the example below, mouse tramdorin 3 cDNAs wereobtained by 5′ and 3′ RACE amplification from Swiss Webster mouse brainRACE-ready cDNA (Ambion), and by amplification of the same mouse braincDNA using primers, as detailed in the example below. The cDNA depictedin FIG. 23A contains the entire tramd3 protein coding region, but doesnot contain the entire 3′ untranslated region.

[0174] Composite mouse tramdorin 2 cDNA (SEQ ID NO: 1) is presented inFIG. 22. As described in the example below, 5′ and 3′ RLM-RACE (Maruyamaet al., Gene 138:171 (1994); Schaefer, Anal Biochem 227:255 (1995)) wereused to isolate mouse tramdorin 2 cDNAs from a mouse testicle cDNAlibrary (Ambion). The absence of a consensus polyadenylation site andthe observation that the poly(A) sequences at the 3′ end are found ingenomic DNA indicate that the 3′ RACE cDNA is not full-length.

IV. Functional Characterization of Tramdorins

[0175] A. Identification of Tramdorin Ligands

[0176] The signal(s) from axons that initiate myelination are unknown.Several molecules that stimulate Schwann cell myelination have beenidentified. Insulin-like growth factor promotes Schwann cellproliferation in conjunction with other growth factors, but in theirabsence, it promotes Schwann cell expression of P0 and myelination(Russell et al., J Neuropathol Exp Neurol 59:575 (2000); Stewart et al.,Eur J Neurosci 8:553 (1996)). Progesterone and glucocorticoids have beenshown to enhance the rate of myelination. However, none of thesemolecules has been shown to be sufficient for triggering myelination bySchwann cells. Studies on regenerating nerves following injury indicatethat axonal contact is required for Schwann cells to myelinate (reviewedin Scherer et al., Axon-Schwann cell interactions in peripheral nerveregeneration. In Jessen, K. R. and Richardson, W. D. (eds.), Glial CellDevelopment, Bios Scientific Publishers, Oxford, [1996] pp. 165-196).However, Schwann cells repressed expression of p75NGFR, and expressedOct-6 and P0 in response to neurons that were separated by a permeablemembrane, suggesting that at least one important axonal signal for thedifferentiation of Schwann cells to a myelinating phenotype is adiffusible molecule (Bolin et al., J Cell Biol 123:237 (1993)). Intheory, axons can control the thickness of myelin sheathes that surroundthem by emanating a single myelinogenic signal which both triggersmyelination, and instructs it to continue as long as the signal ispresent; cessation of the signal terminates myelination (Fraher et al.,J Anat 193:195 (1998)). Based on its homology to the vesicular GABAtransporter, tramdorin protein may function as a transporter of smallmolecules. If tramdorin is required for proper myelination, it maytransduce a signal from axons that promotes myelination, and thereforemay be a crucial reagent in the identification of the myelinogenicsignal itself. Alternatively, tramd1 could interact directly withmolecules that are expressed on the surface of axons, or it could act asa cofactor for other Schwann cell signaling molecule(s). Tramd2 andtramd3 genes may function similarly in the differentiation of other celltypes as well.

[0177] One of skill in the art will understand that there are manymethods that can be used to screen for tramdorin ligands. The presentinvention contemplates that any such assay may be used. By way ofnon-limiting example, one such assay involves identification oftramdorin ligands by the differential import of radiolabelled candidateligands following transient expression of tramdorins in cultured cells.COS-7 cells will be transiently transfected with pCDNA3.1 plasmids(Invitrogen) with or without tramdorin sequences. Following exposure to[³H] amino acids, the differential uptake of the radiolabelled aminoacids will be measured by scintillation counting of cell extracts, asperformed previously for rat LYAAT-1 [Sagne et al., PNSA USA 98:7206(2001)]. Briefly, tramdorin cDNA sequences will be cloned into pcDNA3.1plasmids, which will be introduced into COS-7 cells with Lipofectin(Life Technologies, Grand Island, N.Y.). Transport assays will becarried out 36-48 hours after the beginning of transfection. Followingexposure to [³H] amino acids, cells are lysed in 0.1 N NaOH, and theradioactivity will be measured by scintillation counting in Aquasol(Packard) (see Example below).

[0178] As rat LYAAT-1 is a tramdorin 3 homolog, and Sagne et al. (supra)used the COS-7 transfection and transport assay to successfully identifythe ligand for LYAAT-1, there is compelling evidence that such an assaywill be successful at identifying the ligands of human and mousetramdorins, as well as rat tramdorin 1.

[0179] Another assay contemplated for the identification of tramdorinligands is the expression of tramdorin in Xenopus oocytes. Xenopusoocytes have been used extensively to characterize transport proteins(reviewed in (Miller et al., Biochim Biophys Acta 1465:343 (2000);Theodoulou et al., Methods Mol Biol 49:317 (1995); Wagner et al., CellPhysiol Biochem 10:1 (2000)). In the Xenopus oocyte expression system,oocytes removed from ovaries of adult Xenopus are arrested at the firstmeiotic prophase. Because each oocyte is fairly large and has a largenucleus, it is technically easy to inject foreign DNA into the oocytenucleus or mRNA injected into the cytoplasm. In this case, a synthetictramdorin message is made that possesses a 5′ cap and a poly(A)+ tail,and in which the tramdorin coding sequences are flanked by Xenopusβ-globin 5′ and 3′ untranslated regions. The synthetic message ismicroinjected into the cytoplasm, followed several days later bycharacterization of the expressed protein. Alternatively, tramdoringenes can be microinjected directly into the nuclei of Xenopus oocytes,following cloning into the expression plasmid pMT3 (Swick et al., PNSAUSA 89:1812 (1992)). Tramdorin proteins will be characterized bymeasurement of the rate and extent of uptake of radiolabelled candidateligands relative to mock injected oocytes, as described above in theCOS-7 assay. The large size of the oocytes makes it relatively easy toinsert electrodes into the oocytes in order to measure theelectrophysiological responses (i.e. patch clamp electrophysiology).Thetwo-electrode voltage clamp can be used to measure transport activity inthe presence of ligand(s).

[0180] B. Effect of Overexpression of Tramdorin Proteins on Myelinationby Schwann Cells

[0181] Primary Schwann cells will form myelin when co cultured withneurons; such co cultures can form the basis for an assay of tramdorinfunction during myclination. Achieving myelination by primary Schwanncells cultured with dorsal root ganglion takes two months; two recentlypublished detailed protocols for this procedure will be relied upon(Kleitman et al., Tissue Culture Methods for the Study of Myelination.In Culturing Nerve Cells, Bank et al. (eds.) Cambridge, MA:MIT Press[1998] pp. 545-658; Li, Methods Cell Biol 57:167 (1998)). Proliferationwill be measured by counting Schwann cells. Any effect of tramdorinexpression on the rate of myelination will be measured by any of thefollowing four methods: 1) Sudan black staining; 2) myelin basic protein(MBP) or P0 immunohistochemistry; 3) thin-layer chromatography tomeasure the amounts of myelin-specific lipids (Vitiello et al., JChromatogr 166:637 (1978)); or 4) fluorescent ceramide incorporation(Bilderback et al., J Neurosci Res 49:497 (1997); Chan et al., PNSA USA95:10459 (1998)).

[0182] Briefly, the uterus is removed from female rats that are 14-15days pregnant (plug day is designated as day 0 of pregnancy) in order toobtain embryonic Schwann cells. Thereafter, the embryos are removed fromthe uterus. Using a dissecting microscope, the entire length of spinalcord is exposed. Dorsal root ganglia (DRGs) are removed from both sidesof the spinal cord using forceps. The DRGS are put into culture withcollagenase at 37C for 30-45 minutes, washed and resuspended inHamsF12/DMEM medium (1:1 mixture) supplemented with growth factors. Thecell suspension is then placed in 24-well plates that have been coatedwith natural mouse laminin.

[0183] Postnatal day 0-1 rats are used for the preparation of neonatalrat Schwann cells and the dissociation of DRGs is performed withcollagenase as described above. Adult rat Schwann cells are usuallyprepared from young adult rats 60-90 days old. The dissociation isperformed with a 10% collagenase/dispase solution at 37C for 45 minutes.Human Schwann cells can be prepared from donated organs. The sural nerveis particularly useful. Again, dissociation is achieved with mild enzymetreatment and culturing is done on laminin-coated plates.

[0184] After three days, the cultures on laminin will form a confluentmonolayer of rapidly dividing Schwann cells. The cells can besubcultured every 4 days at a 1:4 split ratio.

[0185] To obtain neurons for coculture with Schwann cells forremyelination, a portion of the dissociated DRGs (after washing butbefore culturing) are placed in a tube with a 5% BSA layer andcentrifuged such that the cloudy cell suspension stays on top of thelayer (and is discarded) and the pellet is recovered, treated with a0.125% trypsin solution, washed and resuspended. The recovered materialcan be further purified on another 5% BSA layer at 1 g. The neurons aregrown in the laminin-coated wells in F12/DMEM supplemented with 5 ng/mlneurotropin 3 and 15% fetal bovine serum.

[0186] To test the effects of tramdorin expression on Schwann cellproliferation, differentiation, and survival, tramdorin proteins will beexpressed in cultured primary Schwann cells using retroviral vectors.Retroviral infection of Schwann cells has several advantages. First, theability to transfect primary Schwann cells with retroviral vectors iswell established, and infected cells can myelinate (Howe et al., JNeurosci Methods 83:133 1998; Owens, Ann NY Acad Sci 633:543 (1991);Owens et al., Development 112:639 (1991); Owens et al., J Cell Biol111:1171 (1990); Owens et al., Neuron 7:565 (1991); Zoidl et al., Embo J14:1122 (1995)). Second, the introduced DNA integrates into the genome,generating a stably transfected cell. To constitutively expressexogenous genes in primary Schwann cells, a dicistronic retroviralsystem, LIRES-GFP, that permits myelination of infected primary Schwanncells (Howe et al., J Neurosci Methods 83:133 (1998)) will be used. TheLIRES-GFP vector has been kindly provided by K. McCarthy. The readingframes will be inserted into the LIRES-GFP vector, after addition ofKozak start sites, and C-terminal HA tags. The pBKS plasmid (Stratagene)has been modified to generate a convenient shuttle vector for addingthese sequences. Retroviruses will be made using the Ecopak ecotropiccell line (Clontech) that permits infection of only rat or mouse cells.After infection of primary Schwann cells with tramdorin retroviruses,the effects of tramdorin expression on Schwann cell proliferation, andon the expression of Oct-6 and myelin P0, will be measured, usingantisera specific for these proteins (Bermingham et al., Gene Develop10: 1751 (1996); Scherer et al., J Neurosci 15:8281 (1995)). Any effectsof its expression in the presence and absence of the putative tramdligand GABA will be measured.

[0187] Myelinating Schwann cell lines may provide an alternative to theuse of primary Schwann cell lines. Two myelinating Schwann cell lineshave been reported. The SCL4.1/F7 cell line was generated by multiplepassaging of primary Schwann cells with intermittent exposure to choleratoxin (Haynes et al., J Neurosci Methods 52:119 (1994)). CR1b4 is aSchwann cell line that was immortalized using a temperature sensitiveoncogene (Thi et al., J Exp Biol 201:851 (1998)).

[0188] C. Generation of Mice That Lack Tramdorin Genes

[0189] To determine the extent to which tramdorin expression is requiredfor peripheral myelination, mice that lack the tramd1 gene (SEQ ID NO:7) will be generated. Two distinct mutations will be made. The firstwill generate a conditional knockout of the tramdorin1 gene (SEQ ID NO:7), in which tramdorin 1 is deleted only in Schwann cells. This approachis based on use of the bacteriophage site-specific recombinase Cre,which recognizes 34 bp loxP sites (reviewed in (Sauer, Methods 14:381(1998)). To generate a mouse in which a gene is deleted in a specifictissue (reviewed in (Marth, J Clin Invest 97:1999 (1996)), the region tobe deleted is flanked by loxP sites. For tramdorin1, exon 2 will beflanked with loxP sites, thereby permitting its specific deletion in thepresence of Cre recombinase. Such a deletion will remove conserved (andtherefore presumably important) sequences, and will cause the remainderof the tramdorin message to be shifted out of frame. Because the twoloxP sites are located in the introns surrounding exon2, the mutationwill have no effect in the absence of Cre recombinase. Such “floxed”mice will be crossed to transgenic mice in which Cre recombinase isexpressed under control of the P0 promoter (Feltri et al., Ann NY AcadSci 883:116 (1999); Giovannini et al., Genes Dev 14:1617 (2000);Yamauchi et al., Dev Biol 212:191 (1999)). Mice that carry both thetransgene, and are homozygous for the floxed tramdorin1 allele will havethe tramdorin gene specifically mutated in Schwann cells. That is tosay, it will take another backcross to the floxed mouse to gethomozygous floxed and the transgene.

[0190] The second mutation will fuse the E. coli LacZ gene in frame totramdorin1 exon 2, thereby truncating the protein and permitting itsexpression to be monitored by β-galactosidase activity.

[0191] D. Screening for Tramd Interacting Proteins

[0192] Although a precise understanding of the mechanism of tramdorinaction is not required for the practice of the present invention, it ishypothesized that tramdorin is required for proper myelination.Tramdorin may transduce a signal from axons that promotes myelination,and therefore may be a crucial reagent in the identification of themyclinogenic signal itself (see Identification of Tramdorin Ligands,above). Alternatively, tramd1 may interact directly with molecules thatare expressed on the surface of axons, or it could act as a cofactor forother Schwann cell signaling molecule(s). Thus, identification ofproteins which interact with tramdorins (especially tramdorin 1) islikely to contribute to an understanding of the myelination process. Anumber of methods are available to detect protein-protein interactions,including but not limited to co-immuoprecipitation, affinitychromatography with immobilized tramdorin 1 protein or fragments orportions of the tramdorin 1 protein, the yeast two-hybrid system andcross-linking. Any suitable method for detection oftramdorin-interacting proteins is contemplated, including the yeast-twohybrid screen and its variations.

[0193] Yeast two-hybrid screening (Fields et al., Nature 340:245 (1989);U.S. Pat. No. 5,667,973, herein incorporated by reference) is animportant approach for identifying protein-protein interactions(reviewed in (Fields et al., Trends Genet 10:286 (1994)). Typically, aprotein of interest, the bait, is fused to the DNA binding domain ofyeast Gal4, or E.coli LexA. A library of targets, or “prey” are fused toa transcriptional activator. The bait and prey fusion proteins arecoexpressed, and if they interact, marker genes, which contain a bindingsite for the DNA-binding domain, are expressed that permit isolation ofplasmids that encode the interacting protein. The marker gene may encodean enzyme or other product that can be readily measured. Such measurableactivity may include the ability of a cell to grow only when the markergene is transcribed, the presence of detectable enzymatic activity (e.g.β-galactosidase encoded by the LacZ gene) only when the marker gene istranscribed, or light release, depending on the type of reporter ormarker gene activated. Commercially available kits for yeast two-hybridscreening are available from Clontech and Stratagene. However, becausethe bait and prey proteins are imported to the nucleus, the standardyeast two-hybrid methodology precludes posttranslational proteinmodifications that may be critical for some protein-proteininteractions. Furthermore, membrane proteins are underrepresented amonginteracting protein pairs that have been found by two-hybrid screening(Uetz et al., Nature 403:623 (2000)). Recently, a new yeast two-hybridsystem, the Ras recruitment system (RRS), has been developed thatutilizes a cytoplasmic interaction to activate a Ras signaling pathway(reviewed in (Broder et al., Curr Biol 8:1121 (1998)). U.S. Pat. No.5,776,689, herein incorporated by reference, also describes the proteinrecruitment system to detect protein-protein interaction at or in aspecific cell compartment. A yeast two hybrid system, CytoTrap, that isbased on RRS methodology, is available from Stratagene. Briefly, thissystem involves the complementation of a temperature-sensitive mutationin the yeast cdc25 gene, the yeast homolog for hSos, by properlocalization of human Sos to the plasma membrane by protein-proteininteraction. A library of target proteins are expressed as fusionproteins with src myristylation sites that anchor the proteins to themembrane. The bait is fused to human Sos, and interaction with targetproteins localizes Sos to the membrane, thereby permitting cell survivaland growth at 37° C. by activation of the Ras signaling pathway. Thus,when the cDNA library and the tramd1-hSos fusion construct aretransformed into the cdc25H yeast strain (Stratagene), the only cellscapable of growing at 37° C. on galactose medium are those that havebeen rescued by a protein-protein interaction recruiting hSos to thecell membrane.

[0194] A reverse RRS technique permits full length transmembrane domainproteins to be used as bait (Hubsman et al., Nucleic Acids Res 29:E18(2001)), has recently been described. This reverse RRS system iscontemplated herein for the isolation of proteins that interact withtramdorins. Full-length tramdorin will be expressed in yeast whereas theprey will be fused to a mutant Ras protein that lacks a membranelocalization signal and stop codon. Those fusion proteins that interactwith tramdorin will be localized to the membrane, thereby permittinggrowth at restrictive temperature of yeast that carry thetemperature-sensitive Cdc25-2 mutation.

[0195] Although a precise understanding of the localization of tramdorinis not required for the practice of the invention, it is possible thattramdorin may be localized to lysosomes. If this is the case, then therecruitment of Ras activity to lysosomes may not properly activate rassignaling pathways using the reverse RRS technique. Therefore,conventional yeast two-hybrid screening for tramdorin-interactingproteins is also contemplated. The following two baits will be used inthe conventional screening:

[0196] (i) The N-terminal 51 Amino Acids of Mouse Tramdorin1:

[0197] MSVTKSARSPQVATPLNLDLPESAKKLQSQDPSPANGTSSESSKKTKGITG (SEQ ID NO:15)

[0198] This is encoded by the DNA sequence: (SEQ ID NO:45)      ATG TCTGTG ACC AAG AGT GCC AGG AGT CCG CAG GTA GCC ACC CCT CTC AAT CTG GAC CTTCCT GAG AGT GCC AAG AAG CTG CAG AGC CAG GATCCC AGT CCA GCG AAT GGG ACCTCT TCA GAG TCA TCA AAG AAG ACC AAG GGC ATA ACC GGG

[0199] (ii) The First Putative Intracellular Loop of Mouse Tramdorin 1,Which Consists of the Following 39 Amino Acid Sequence:

[0200] RCAQRFCHRLNKPFMDYGDTVMHGLAFSPNAWLQNHAHW (SEQ ID NO: 16)

[0201] This is encoded by the DNA sequence: (SEQ ID NO:46)      AGA TGTGCC CAG CGC TTC TGT CAC AGA CTG AAC AAG CCT TTC ATG GAC TAT GGG GAC ACAGTG ATG CAC GGA CTG GCT TTC AGT CCC AAT GCC TGG CTG CAA AAC CAC GCC CACTGG

[0202] These are the largest amino acid sequences within the tramdorin 1protein that do not contain predicted transmembrane domains. TheN-terminal 51 amino acids have proven to be toxic when expressed in E.coli. If the same holds true in yeast, the intracellular loop bait willbe used. DNA sequences that encode the baits will be cloned into pSos(Stratagene) for RRS two hybrid screening, or pBD-Gal4 (Stratagene) forconventional Gal4-based yeast two hybrid screening, or pLexA (Clontech)for conventional yeast two-hybrid screening based on LexA.

[0203] For both the RRS and conventional yeast two-hybrid screening thelibrary of prey interacting proteins will be derived from an adult mousesciatic nerve cDNA library that will be constructed as follows: Sciaticnerves have been collected from adult male FVB/NJ mice (these areavailable free of charge). Total RNA has been isolated from the sciaticnerves by CsCl₂ gradient centrifugation (Chirgwin et al., Biochem18:5294 (1979)). cDNA will be synthesized from poly A⁺ purified RNAusing Superscript cDNA synthesis kit (Gibco-BRL), and cloned into thepYes2 (Invitrogen) for reverse RRS screening, the yeast two hybridlibrary plasmid pMyr (Stratagene) for RRS screening, or pAD-Gal4-2.1(Stratagene) for conventional Gal4-based yeast two hybrid screening, orpB42AD (Clontech) for conventional yeast two hybrid screening based onLexA.

[0204] By way of example of selection and screening for protein-proteininteractions, in the Stratagene system, as described by themanufacturer, yeast (strain YRG-2; Matα, ura3-52, his3-200, ade2-101,lys2-801, trp-1901, leu2-3 11, gal4-542, gal80-538, LYS2::UAS_(GAL1)-TATA_(GAL1)-HIS3 URA3::UAS_(GAL4 17mers(×3))-TATA_(CYCI)-lacZ)can be transformed with the pBD-GAL4/protein of interest fragment (i.e.tramd 1 in one embodiment contemplated herein) vector and the mousesciatic nerve cDNA library contained in the pAD-GAL4-2.1 vector andselected on plates that lack leucine (selection for the pAD-GAL4-2.1vector) and tryptophan (selection for the pBD-GAL4 vector), andscreening can be done by virtue of the histidine and LacZ reporter genesin YRG-2.

[0205] E. Analysis of Tramdorin Gene Expression Patterns

[0206] 1. Tissue Preparation

[0207] The spatial and temporal expression patterns of the tramdoringenes were identified by in situ hybridization using radiolabelled RNAprobes that specifically recognize individual tramdorin genes. For insitu hybridization, tissue sections were obtained as follows. Mice wereanesthetized and perfused with 4% paraformaldehyde or formalin.Hindquarters were dissected to expose the nerve, then stored at 4° C. in4% paraformaldehyde or formalin, then processed as follows. Tissues werefrozen in a 1:1 mixture of OCT and Aquamount, and sectioned at 20microns.

[0208] 2. In Situ Hybridization

[0209] In situ hybridization was performed as described by Simmons etal., J. Histotechnology 12:169 (1989). Darkfield micrographs of adjacentsections of P0 mouse leg (prepared as described in the previous section)containing femur and fat pad were hybridized with ³⁵S labeled probesspecific for tramd1, tramd2 or tramd3. As seen in FIG. 31, the tramd1probe hybridized to fat pads, and to bone marrow, but not to growthplate. No hybridization to either tissue was detected for tramd2 ortramd3. While it is not intended that the present invention be limitedto any specific mechanism, these data suggest a role for glycinesignaling in the differentiation of bone and fatty tissue. The probesconsist of 343 nt between nucleotides 1549 and 1892 of mouse tramdorin1cDNA cDNA AF512429 ; 347 nt between nucleotides 2 and 349 of mousetramdorin 2 cDNA, and 323 nt between nucleotides 1506 and 1829 of mousetramdorin 3 cDNA. Scale bar: 500 microns.

[0210] It has been shown that that bone has NMDA (N-methyl-D-aspartate)receptors. See, Gu et al., “Expression Of Functional MetabotropicGlutamate Receptors In Primary Cultured Rat Osteoblasts”, J. Biol.Chem., Vol. 275, pp. 34252-34259 (2000). In one embodiment of thepresent invention, therefore, it is contemplated that tramdorins may beused as a marker in bone. In another embodiment, it is contemplated thattramdorins may be administered to a subject to modulate theproliferation and differentiation of bone cells (including, but notlimited to, osteoblasts and osteoclasts).

[0211] In one embodiment of the present invention, it is contemplatedthat tramdorins may be used as a marker in adipose tissue. In anotherembodiment, it is contemplated that tramdorins may be administered to asubject to modulate the proliferation of fat cells (including, but notlimited to, adipocytes) and the incorporation of glycine into fat cells.

[0212] 3. Preparation Of Probes Specific For Tramd1, Tramd2 and Tramd3

[0213] The probes used in the in situ hybridization reactions, asdepicted in FIG. 31, were prepared as follows.

[0214] Mouse tramdorin1 probe (SEQ ID NO: 71). Mouse tramd1 sequences,shown in bold, were amplified by PCR, and cloned into pCRII(Invitrogen). The insert was excised using EcoRi and cloned into theEcoRi site of pBKSII+ (Stratagene). EcoRi sites are boxed. The probe istranscribed from the T7 promoter, shown in italics, using T7 polymerase,using plasmid DNA that has been linearized at the HindIII site, which isunderlined. The mouse tramdorin1 sequence consists of 343 bp betweennucleotides 1549 and 1892 of AF512429, shown in antisense orientation.

[0215] Mouse tramdorin2 probe (SEQ ID NO: 72). 347 bp of mouse tramdorin2 sequences between nucleotides 2 and 349, shown in bold, were amplifiedby PCR, and cloned into pCRII (Invitrogen) They are shown in antisenseorientation. EcoRI sites are boxed. The probe was transcribed from theT7 promoter, shown in italics, using T7 polymerase, using plasmid DNAthat has been linearized at the BamHI site, which is underlined.

[0216] Mouse tramdorin3 probe (SEQ ID NO; 73). 323 bp of mousetramdorin3 sequences between nucleotides 1506 and 1829 of mousetramdorin3 composite cDNA 50+61-45+76, shown in bold, were amplified byPCR, and cloned into pCRII (Invitrogen). They are shown in antisenseorientation. EcoRI sites are boxed. The probe was transcribed from theT7 promoter, shown in italics, using T7 polymerase, using plasmid DNAthat has been linearized at the BamHI site, which is underlined.

[0217] 4. Tramdorin Antiserum

[0218] i. Preparation

[0219] Antisera against tramdorin proteins permit their subcellularlocalization to be determined by immunohistochemistry and confocalmicroscopy. Antisera were raised in two rabbits against the mousetramdorin 1 C-terminal peptide CGTYQALDELIKSGNSPA (SEQ ID NO: 47). Theantisera were affinity purified using a Sulfolink column (Pierce) thatcontained covalently bound peptide. However, the antisera showedextremely weak signals on tissue sections under a variety of conditions.To obtain antisera that provide better signal on tissue sections, asynthetic peptide: ESAKKLQSQDPSPANGTSC (SEQ ID NO: 74), containing aminoacids 22-39 near the N-terminus of tramdorin1, was coupled to KLH andinjected into two rabbits, one of which generated useful antiserum. Itshould be noted that the C-terminal cystine is used for linkage to KLH.

[0220] In other embodiments alternative approaches, such as expressionthe N-terminus as a GST fusion protein or to generate antisera againstsynthetic peptide(s) that correspond to this region, may be used.

[0221] While the cloning of this tramdorin 1 N-terminus expressionplasmid has proven to be non-trivial, there are viable alternativestrategies to obtain antisera against the N-terminus of mousetramdorin 1. By way of non-limiting example, the N-terminus can beexpressed as a GST-fusion protein, which permits purification of thefusion on an affinity column. The GST portion of the fusion can beremoved before injection into animals for antisera generation, or theentire fusion can be used to generate antiserum, as long as theantiserum is affinity cleared of any GST-reactive antibodies before usein localization studies. Another strategy is to generate antiseraagainst synthetic peptide(s) that correspond to this region. Suchsynthetic peptides can be chemically prepared by methods well known inthe art, and do not require expression in a host organism.

[0222] The present invention provides isolated antibodies. An antibodyagainst a protein of the present invention (i.e. a tramdorin protein orfragment or portion of a tramdorin protein) may be any monoclonal orpolyclonal antibody, as long as it can recognize the protein ofinterest. Antibodies can be produced by using a protein, or fragmentthereof, or synthetic peptide, of the present invention as the antigenaccording to a conventional antibody or antiserum preparation process.

[0223] The present invention contemplates the use of both monoclonal andpolyclonal antibodies. Any suitable method may be used to generate theantibodies used in the methods and compositions of the presentinvention, including but not limited to, those disclosed herein. Forexample, for preparation of a monoclonal antibody, protein, as such, ortogether with a suitable carrier or diluent is administered to an animal(e.g., a mammal) under conditions that permit the production ofantibodies. For enhancing the antibody production capability, completeor incomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total, about 2times to about 10 times. Animals suitable for use in such methodsinclude, but are not limited to, primates, rabbits, dogs, guinea pigs,mice, rats, sheep, goats, etc.

[0224] For preparing monoclonal antibody-producing cells, an individualanimal whose antibody titer has been confirmed (e.g., a mouse) isselected, and 2 days to 5 days after the final immunization, its spleenor lymph node is harvested and antibody-producing cells containedtherein are fused with myeloma cells to prepare the desired monoclonalantibody producer hybridoma. Measurement of the antibody titer inantiserum can be carried out, for example, by reacting the labeledprotein, as described hereinafter and antiserum and then measuring theactivity of the labeling agent bound to the antibody. The cell fusioncan be carried out according to known methods, for example, the methoddescribed by Koehler and Milstein [Nature 256:495 (1975)]. As a fusionpromoter, for example, polyethylene glycol (PEG) or Sendai virus (HVJ),preferably PEG, is used.

[0225] Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1 and thelike. The proportion of the number of antibody producer cells (spleencells) and the number of myeloma cells to be used is preferably about1:1 to about 20:1. PEG (preferably PEG 1000-PEG 6000) is preferablyadded in concentration of about 10% to about 80%. Cell fusion can becarried out efficiently by incubating a mixture of both cells at about20° C. to about 40° C., preferably about 30° C. to about 37° C. forabout 1 minute to 10 minutes.

[0226] Various methods may be used for screening for a hybridomaproducing the antibody (e.g., against a tramdorin protein or portionthereof). For example, where a supernatant of the hybridoma is added toa solid phase (e.g., microplate) to which protein is adsorbed directlyor together with a carrier, and then an anti-immunoglobulin antibody (ifmouse cells are used in cell fusion, anti-mouse immunoglobulin antibodyis used) or Protein A labeled with a radioactive substance or an enzymeis added to detect the monoclonal antibody against the protein bound tothe solid phase. Alternately, a supernatant of the hybridoma is added toa solid phase to which an anti-immunoglobulin antibody or Protein A isadsorbed and then the protein labeled with a radioactive substance or anenzyme is added to detect the monoclonal antibody against the proteinbound to the solid phase.

[0227] Selection of the monoclonal antibody can be carried out accordingto any known method or its modification. Normally, a medium for animalcells to which HAT (hypoxanthine, aminopterin, thymidine) are added isemployed. Any selection and growth medium can be employed as long as thehybridoma can grow. For example, RPMI 1640 medium containing 1% to 20%,preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to10% fetal bovine serum, a serum free medium for cultivation of ahybridoma (SFM-101, Nissui Seiyaku) and the like can be used. Normally,the cultivation is carried out at 20° C. to 40° C., preferably 37° C.for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5%CO₂ gas. The antibody titer of the supernatant of a hybridoma culturecan be measured according to the same manner as described above withrespect to the antibody titer of the anti-protein in the antiserum.

[0228] Separation and purification of a monoclonal antibody (e.g.,against tramdorin or a fragment of tramdorin) can be carried outaccording to the same manner as those of conventional polyclonalantibodies such as separation and purification of immunoglobulins, forexample, salting-out, alcoholic precipitation, isoelectric pointprecipitation, electrophoresis, adsorption and desorption with ionexchangers (e.g., DEAE), ultracentrifugation, gel filtration, or aspecific purification method wherein only an antibody is collected withan active adsorbent such as an antigen-binding solid phase, Protein A orProtein G and dissociating the binding to obtain the antibody.

[0229] Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigen against theprotein) and a carrier protein is prepared and an animal is immunized bythe complex according to the same manner as that described with respectto the above monoclonal antibody preparation. A material containing theantibody against is recovered from the immunized animal and the antibodyis separated and purified.

[0230] As to the complex of the immunogen and the carrier protein to beused for immunization of an animal, any carrier protein and any mixingproportion of the carrier and a hapten can be employed as long as anantibody against the hapten, which is crosslinked on the carrier andused for immunization, is produced efficiently. For example, bovineserum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. maybe coupled to an hapten in a weight ratio of about 0.1 part to about 20parts, preferably, about 1 part to about 5 parts per 1 part of thehapten.

[0231] In addition, various condensing agents can be used for couplingof a hapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide activated ester, activated ester reagents containing thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsthe antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times.

[0232] The polyclonal antibody is recovered from blood, ascites and thelike, of an animal immunized by the above method. The antibody titer inthe antiserum can be measured according to the same manner as thatdescribed above with respect to the supernatant of the hybridomaculture. Separation and purification of the antibody can be carried outaccording to the same separation and purification method ofimmunoglobulin as that described with respect to the above monoclonalantibody.

[0233] The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, tramdorin can be used as theimmunogen. Further, fragments of the protein may be used, including butnot limited to the amino terminus of tramdorin. Fragments may beobtained by any methods including, but not limited to expressing afragment of the gene, enzymatic processing of the protein, chemicalsynthesis, and the like.

V. Tramdorins and Parathyroid Tumors

[0234] As noted above, a fourth human tramdorin-related sequence,tramdL, is defined by the EST cDNA 1388139 (SEQ ID NO: 23) (FIGS. 8D and12). tramdL contains only sequences that are homologous to exon 10 oftramdorin1. This cDNA is derived from a parathyroid adenoma, and isunusual in that its 5′ end consists of sequences derived from a memberof the STE20 family of kinases, STK4/Mst-1/Krs-2 (FIG. 12A; Creasy etal., J Biol Chem 270:21695 (1995); Taylor et al., PNSA USA 93:10099(1996)), located on human chromosome 20. The STK4/Mst-1/Krs-2 cDNAfragment is fused to 3′ sequences of tramdL (FIG. 12B), located onchromosome 5. Comparison of the genomic sequences for STK4/Mst-1/Krs-2and tramdL reveal that the fusion site corresponds to a 5′ splice sitein the STK4/Mst-1/Krs-2 gene, and a 3′ splice site in tramdL (FIG. 12C).Such a fusion is unlikely to be an artifact of cDNA synthesis. While anunderstanding of the precise mechanism is not necessary to the practiceof the invention, and noting that the invention is not to be limited toany particular mechanism, this observation raises the possibility thattramdL resides near a parathyroid tumor translocation breakpoint,between chromosomes 20 and 5, that results in expression of aSTK4/Mst-1/Krs-2-tramdorinL fusion protein. STK4/Mst-1/Krs-2 contains aC-terminal inhibitory domain (Creasy et al., J Biol Chem 271:21049(1996)) that is cleaved by Caspase-3 to generate a cleavage product,p36, with kinase activity (Graves et al., Embo J 17:2224 (1998); Lee etal., Oncogene 16:3029 (1998); Watabe et al., J Biol Chem 275:8766(2000)). The STK4/Mst-1/Krs-2-tramdorin1 fusion protein would replaceSTK4/Mst-1/Krs-2 C-terminal sequences with a C-terminal transmembranedomain from tramdL (FIG. 12D). Based on the activity of C-terminaldeletions of STK4/Mst-1/Krs-2 (Creasy et al., J Biol Chem 271:21049(1996)), the fusion protein may retain kinase activity, and mayrepresent an important aspect of the etiology of parathyroid tumors.

[0235] In one embodiment of the present invention, it is contemplatedthat tramdorins may be used as a marker in bone. In another embodimentof the present invention, it is contemplated that tramdorins may beadministered to a subject to modulate the proliferation anddifferentiation of bone cells (including, but not limited to,osteoblasts and osteoclasts).

[0236] A. Parathyroid Adenoma Background

[0237] Primary hyperparathyroidism is a disorder of the parathyroidglands. Most people with this disorder have one or more enlarged,overactive parathyroid glands that secrete too much parathyroid hormone.The parathyroid glands are four pea-sized glands located on the thyroidgland in the neck. The parathyroid glands secrete parathyroid hormone(PTH), a hormone that helps maintain the correct balance of calcium andphosphorus in the body. PTH regulates the release of calcium from bone,absorption of calcium in the intestine, and excretion of calcium in theurine. When the amount of calcium in the blood falls too low, theparathyroid glands secrete just enough PTH to restore the balance.

[0238] If the glands secrete too much hormone, as inhyperparathyroidism, the balance is disrupted: blood calcium rises. Thiscondition of excessive calcium in the blood, called hypercalcemia, iswhat usually signals the doctor that something may be wrong with theparathyroid glands. In 85 percent of people with this disorder, a benigntumor (an adenoma) has formed on one of the parathyroid glands, causingit to become overactive. The excess PTH triggers the release of too muchcalcium into the bloodstream. The bones may lose calcium, and too muchcalcium may be absorbed from food. The levels of calcium may increase inthe urine, causing kidney stones. PTH also acts to lower the bloodphosphorus levels by increasing excretion of phosphorus in the urine.

[0239] In the United States, about 100,000 people develophyperparathyroidism each year. Women outnumber men by 2 to 1, and riskincreases with age. In women 60 years of age and older, 2 out of 1,000will get hyperparathyroidism. Primary hyperparathyroidism is found in upto 2.1% of postmenopausal women (Lundgren et al., Surgery 121:287(1997)). A person with hyperparathyroidism may have severe symptoms,subtle symptoms, or no symptoms at all. Routine blood tests that screenfor high calcium levels are alerting doctors to people who, thoughsymptom-free, have mild forms of the disorder. When symptoms do appear,they are often mild and nonspecific, such as feelings of weakness andfatigue, depression, or aches and pains. With more severe disease, aperson may have a loss of appetite, nausea, vomiting, constipation,confusion or impaired thinking and memory, and increased thirst andurination. Patients may have thinning of the bones without symptoms, butwith risk of fractures. Increased calcium and phosphorus excretion inthe urine may cause kidney stones. Subjects with hyperparathyroidism maybe more likely to develop peptic ulcers, high blood pressure, andpancreatitis.

[0240] Hyperparathyroidism is diagnosed when tests show that bloodlevels of calcium as well as parathyroid hormone are too high. Once thediagnosis is established, other tests may be done to assesscomplications. Because high PTH levels can cause bones to weaken fromcalcium loss, a measurement of bone density may be done to assess boneloss and the risk of fractures. Abdominal radiographs may reveal thepresence of kidney stones and a 24-hour urine collection may provideinformation on kidney damage and the risk of stone formation.

[0241] At the present time, surgery to remove the enlarged gland is theonly treatment for the disorder. While surgery is an effective cure in95% of cases, by nature, surgery is invasive and carries risks ofcomplications, including but not limited to post-surgical infection.Some patients who have mild disease may not need immediate treatment.Such patients are subject to long-term monitoring which includes regulartesting of calcium levels, bone mass and kidney function. Such subjectsmust avoid certain medications (such as diuretics such as the thiazides)and consult a physician in cases of immobilization or gastrointestinaldisease with vomiting and diarrhea, which can cause calcium levels torise. Thus, a non-invasive therapeutic, an animal model to test suchtherapeutics, and a diagnostic test to determine the presence of certaingenomic rearrangements in parathyroid adenomas to direct the therapeuticregimen would all be useful.

[0242] At the present time, the underlying molecular genetic pathologyof parathyroid adenomas is not fully understood. In a subset ofparathyroid adenomas, a tumor-specific DNA rearrangement (apericentromeric inversion, inv(11)(p15;q13)) which results in the fusionof the cyclin D1 gene (CCND1) with the parathyroid hormone regulatoryregion is found. Immunohistochemistry has shown that cyclin D1 isoverexpressed in 20-40% of parathyroid adenomas. However, other (noncyclin dependent) mechanisms may account for parathyroid cellproliferation in the remaining cases (see for example, Mallya et al.,Frontiers in Bioscience 5:d367-371 (2000)). Mallya et al. (supra) alsonote that mice which harbor a transgene in which the cyclin DI gene isplaced under the control of the parathyroid hormone (PTH) regulatoryregion (to mimic the chromosome 11 rearrangement and resultant cyclin D1overexpression in the human tumors) develop hyperparathyroidism by theage of six months, as evidenced by parathyroid enlargement and increasedserum calcium and parathyroid hormone levels. Thus, tissue specificexpression of cyclin D1 does induce parathyroid cell proliferationresulting in hyperparathyroidism. However, this etiology is only likelyto account for 20-40% of parathyroid adenomas.

[0243] B. Tramdorins in Parathyroid Adenomas

[0244] While not limited to any particular mechanism, and with the notethat an understanding of the underlying mechanism is not necessary tothe practice of the invention, the identification of a putativeSTK4/Mst-1/Krs-2-tramdorinL fusion gene that is derived from parathyroidadenomas suggests that a membrane-bound, constitutively active kinasemay function in the etiology of these tumors. Parathyroid adenoma DNAhas been examined for allelic loss (Tahara et al., Cancer Res 56:599(1996)), or chromosome gain or loss (Palanisamy et al., J ClinEndocrinol Metab 83:1766 (1998)), as an approach to identifyingparathyroid tumor suppressor genes, but these studies would notnecessarily detect translocations. The putativeSTK4/Mst-1/Krs-2-tramdorinL fusion gene is suggestive of a t(20;5)translocation in at least one parathyroid adenoma. STK4/Mst-1/Krs-2 hasbeen shown to be activated by apoptotic signals (Graves et al., Embo J17:2224 (1998); Lee et al., Oncogene 16:3029 (1998); Watabe et al., JBiol Chem 275:8766 (2000)), and its active proteolytic fragment has beenfound in several tumor cell lines (Watabe et al., J Biol Chem 275:8766(2000), but its physiological role is not understood. If the putativetruncated, membrane-bound form of the kinase contributes to the etiologyof parathyroid adenomas, it may provide avenues toward new therapies fora disorder that will increase in frequency in the United States as theaverage age of Americans increases.

[0245] A number of means are contemplated for determining the role ofthe putative STK4/Mst-1/Krs-2-tramdorinL fusion gene in parathyroidadenomas, and the invention is not intended to be limited to anyparticular means of evaluating the putative STK4/Mst-1/Krs-2-tramdorinLfusion gene in parathyroid adenomas. For example, DNA-based assays arecontemplated, wherein the fusion is detected by PCR amplification ofcDNA from parathyroid adenomas using primers corresponding to the tramdLportion of the fusion and the STK4/Mst-1/Krs-2 portion of the fusion,such that amplification will occur across the fusion junction if thefusion is present, but no amplification will occur in the absence of thefusion. RNA will be isolated from several independent parathyroidadenomas, as well as other parathyroid tumors, and RT-PCR will becarried out using primers that correspond to tramdL sequences. The cDNAsthat will be obtained from these tissues will determine if the fusiontranscript is present in parathyroid tumor tissue. Limited parathyroidadenoma tissue is available from Research Genetics. Additional sampleswill be obtained from tissue banks, or other sources, such as localphysicians. Primers that can be used to detect the fusion transcriptare:

[0246] 5′ TGCCAAAGGAGTGTCAATACTGCG 3′ (STK4/Mst-1/Krs-1) (SEQ ID NO: 48)

[0247] 5′ TCCCCACCAGAAATCCCACAAAGC 3′ (tramdL) (SEQ ID NO: 49)

[0248] These primers have an optimal annealing temperature of 59° C.,and will amplify a 494 bp product from STK4/Mst-1/Krs-1-tramdL fusioncDNAs. Therefore they may also be used for diagnosis of translocationsthat produce STK4/Mst-1/Krs-1-tramdL fusion transcripts. If fusion cDNAsare detected, primers that correspond to the initiation ATG codon ofSTK4/Mst-1/Krs-1, and the termination TAG codon of tramdL, or sequences3′ to it, will be used for RT-PCR to amplify the entireSTK4/Mst-1/Krs-1-tramdL coding sequence for expression studies.

[0249] Molecular cytogenetic analysis, such as FISH, is alsocontemplated for detection of the presence of theSTK4/Mst-1/Krs-2-tramdL fusion in cells harvested from parathyroidtumors. Bacterial artificial chromosomes (BACs) with sequencescorresponding to tramdL genomic sequences will be obtained, as will BACswith inserts corresponding to the genomic sequence of STK4/Mst-1/Krs-2.The tramdL and STK4/Mst-1/Krs-2 BACS will be differentially labeled. Forexample, the tramdL BAC can be labeled with a marker which permitsdetection of a green fluorescent signal, such as by direct incorporationof fluorescein or FITC, or by incorporation of biotin, which can bedetected by FITC-conjugated avidin. The STK4/Mst-1/Krs-2 BAC can belabeled with a marker which permits detection of a red fluorescentsignal, such as by direct incorporation of TRITC or by incorporation ofdigoxigenin, which can be detected by rhodamine-conjugatedanti-digoxigenin antibodies. The labeled probes are hybridized to acytogenetic preparation of an isolated parathyroid tumor, or culturedcells derived from isolated parathyroid tumors. As a normal control,normal parathyroid tissue (or cultured cells) or peripheral blood cellscan be used. The probes can be prepared by a number of methods,including PCR amplification and nick translation. Suitable nicktranslation protocols are provided in Human Chromosomes, Principles andTechniques (2E), Verma and Babu (Ed.) Chapter 6 (pp. 185-231) (1995) andLichter and Ried “Molecular Analysis of Chromosome Aberrations. In situHybridization” pp. 449-478 in Methods in Molecular Biology, Vol. 29:Chromosome Analysis Protocols. Edited by J. R. Godsen. (1994). Followinghybridization, washing and detection steps, (as described in Verma andBabu and Lichter and Ried, supra) the specimens are counter-stained with4′,6-diamidino-2-phenyl indole dihydrochloride (DAPI) (to permitvisualization of the nuclei and metaphase chromosomes in blue), andobserved and imaged on a fluorescence microscope equipped with a mercuryor xenon lamp and the appropriate filter sets. In the normal controls,the expected result on an interphase nucleus is two green spots (or twoclosely juxtaposed “doublets” in nuclei that have replicated their DNA),corresponding to the two tramdL alleles on chromosome 4, and two redspots (or two closely juxtaposed “doublets” in nuclei that havereplicated their DNA), corresponding to the two STK4/Mst-1/Krs-2 alleleson chromosome 20. In parathyroid adenomas with a STK4/Mst-1/Krs-2-tramdLfusion, the expected result (in a diploid or near-diploid cell) is asingle green spot (or doublet), corresponding to the tramdL allele thatis not involved in the fusion, a single red spot (or doublet),corresponding to the STK4/Mst-1/Krs-2 allele that is not involved in thefusion, and a yellowish-orange “fusion spot” resulting from the nearsuper-imposed hybridization of the green tramdL probe and the redSTK4/Mst-1/Krs-2 probe hybridizing to the translocation-derivedSTK4/Mst-1/Krs-2-tramdL fusion.

[0250] The detection of “fusion” FISH signals is well known in the artas a means to detect translocations, for example, in the detection ofthe BCR/ABL rearrangement that fuses the ABL proto-oncogene onchromosome 9 to the BCR (breakpoint cluster region) gene on chromosome22 and which is frequently present in chronic myelogenous leukemia (CML)(see, for example, “Cellular Genomic Products for HematopoieticDisorders”, available from Vysis, Inc. Downers Grove, Ill.,http://www.vysis.com). Using dual-color FISH, the proportion ofparathyroid tumors harboring a STK4/Mst-1/Krs-2-tramdL fusion can bedetermined.

[0251] It is also possible, and known in the art to use “chromosomepainting” probes to detect translocations in metaphase spreads (see forexample, Speicher et al., Nat Genet 12:368-375, (1996)). Thus, a wholechromosome paint probe to chromosome 5, and a whole chromosome paintprobe to chromosome 20 can be hybridized to metaphase chromosome spreadsfrom parathyroid tumors. Differentially labeled painting probes arecommercially available, for example WCP® 20 Spectrum Green™ from Vysis(Downers Grove, Ill.) is a probe which hybridizes to both arms and thecentromere of chromosome 20. WCP 5® Spectrum Orange™ from Vysis (DownersGrove, Ill.) hybridizes to both arms and the centromere of chromosome 5.Hybridization of these probes to parathyroid adenomas metaphase spreads(according to the manufacturer's instructions) will reveal the presenceof a translocation between chromosome 5 and chromosome 20, in the formof a two-color chromosome. In order to evaluate other rearrangements,spectral karyotyping (SKY) or multi-fluor FISH (Speicher et al., supra;Schrock et al., Science 273:494 (1996)) may be carried out. In thesemethods, a probe “cocktail” with every chromosome labeled with a uniquecombination of dyes is hybridized to metaphase preparations. Followingwashing and detection steps, the hybridized and counterstainedpreparations are imaged on a spectral imaging workstation (for SKY),which determines the spectrum (and hence the dye combination) at eachpoint in the image, allowing unambiguous identification of complexchromosome rearrangements. Thus, any rearrangements involving chromosome5 (and tramdL sequences) and chromosome 20 (and STK4/Mst-1/Krs-2sequences) can be identified.

[0252] Although an understanding of the mechanism underlying theinvention is not necessary to the practice of the invention, it ishypothesized that the STK4/Mst-1/Krs-2-tramdL fusion may be involved inthe proliferation of parathyroid tumors, including but not limited toparathyroid adenomas. In order to investigate this role and create ananimal model which can be used to investigate therapeutic regimens forparathyroid tumors harboring a STK4/Mst-1/Krs-2-tramdL fusion,transgenic mice in which the STK4/Mst-1/Krs-2-tramdL fusion construct(FIG. 26) (SEQ ID NO: 38) is expressed under control of a promoter thatis active in the parathyroid gland are contemplated. Gcm2 is atranscription factor that is expressed primarily, and perhapsexclusively, in parathyroid tissue [Kim et al., PNSA USA 95: 12364(1998)]. Mice will be generated in which the Gcm2 promoter controlsexpression of the STK4/Mst-1/Krs-2-tramdL fusion gene. These mice willbe generated by “knock-in” targeted recombination in embryonic stemcells (ES cells).

[0253] The STK4/Mst-1/Krs-2-tramdL fusion protein (SEQ ID NO: 44) willbe expressed in mice using 5.2kb of the human Pth promoter region. Whenoverexpressed in parathyroid gland using this DNA fragment, the cyclinD1 gene causes primary hyperparathyroidism in mice (Imanishi, Y.,Hosokawa, Y., Yoshimoto, K., Schipani, E., Mallya, S., Papanikolaou, A.,Kifor, O., Tokura, T., Sablosky, M., Ledgard, F, Gronowicz, G, Wang, T.C., Schmidt, E. V., Hall, C., Brown, E. M., Bronson, R., and Arnold, A.2001. Primary hyperparathyroidism caused by parathyroid-targetedoverexpression of cyclin D1 in transgenic mice. Journal of ClinicalInvestigation 107: 1093-1102.)

[0254] Alternatively, the Gcm2 coding regions will is replaced with theSTK4/Mst-1/Krs-2-tramdL fusion gene. The Gcm2 gene has been inactivatedin mice [Gunther et al., Nature 406:199-203, (2000)], indicating thatmice that carry a single knock-in allele will not be affected by theabsence of a single Gcm2 allele. In an alternative embodiment, a“knock-in” of the STK4/Mst-1/Krs-2 into the parathyroid hormone locus iscontemplated.

[0255] Parathyroid-specific expression of the STK4/Mst-1/Krs-2-tramdLfusion is expected to result in hyperparathyroidism (which can bedetected by parathyroid enlargement and increased serum levels ofcalcium and parathyroid hormone), if, as hypothesized, theSTK4/Mst-1/Krs-2-tramdL fusion plays a role in parathyroid tumors. Suchmice can then be used in evaluation of therapeutics which may be usefulin the management of parathyroid tumors harboring theSTK4/Mst-1/Krs-2-tramdL fusion in human subjects

VI. Tramdorins and Peripheral Neuropathies

[0256] As noted above, the human tramdorins map to chromosome 5q31-33.An autosomal recessive Charcot-Marie-Tooth Syndrome demyelinatingneuropathy has been mapped to this region [Guilbot et al., Ann NY AcadSci 883:56 (1999a); Guilbot et al., Eur J Hum Genet 7:849 (1999b);LeGuem et al., Hum Mol Genet 5:1685 (1996)), suggesting that a humantramdorin could be a candidate peripheral neuropathy disease gene. Asnoted above, a crucial function of Oct-6 in myelination may beactivating expression tramdorin 1, a gene that encodes a noveltransmembrane domain protein. Of the six target genes for Oct-6identified thus far, it is the only one that resides near a candidateperipheral neuropathy locus. A search for mutation in families thatsegregate the chromosome 5q32 peripheral neuropathy (Guilbot et al., AnnNY Acad Sci 883:56 (1999a); Guilbot et al., Eur J Hum Genet 7:849(1999b); LeGuem et al., Hum Mol Genet 5:1685 (1996)) is underway. Thus,by focusing on the tramdorins, the target area to look fordisease-associated mutations is significantly reduced. If a consistentdisease-associated mutation in a tramdorin gene can be identified, thatgene can be used in a molecular diagnostic assay, and may alsocontribute to potential therapies. For example, the identification ofthe disease-associated mutation in the cystic fibrosis transmembraneconductance regulator (CFTR) gene enabled the development of adiagnostic genetic test, an animal model, and potential gene therapies(see OMIM entry *602421,http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?602421 and Chapter3 of “Human Genetics, A Problem-based Approach”, Bruce Korf, BlackwellScience, 1996 for a summary), and a similar outcome is expected should aperipheral neuropathy-associated mutation in a tramdorin be identified.

VII. Remyelination by Schwann Cells in the CNS Following Disease orInjury

[0257] Remyelination of axons in the central nervous system (CNS) bySchwann cells has been explored extensively as a possible treatment fordemyelinating diseases such as multiple sclerosis, and as a treatmentfor nerve injury (reviewed in (Duncan et al., J Anat 190:35 (1997);Scolding, Philos Trans R Soc Lond B Biol Sci 354:1711 (1999); Scoldinget al., Baillieres Clin Neurol 6:525 (1997)). MS affects 1/1000Americans, and in people of northern European descent, the frequency is1/800 (Lynch et al., Dis Mon 42:1-55 (1996)); it represents asignificant public health problem in the United States and Europe. Anunderstanding of the genes that regulate myelination by Schwann cells iscrucial for optimizing their use in remyelination in the CNS. Thetramdorin gene may play a crucial role in regulating myelination for thefollowing reasons: First, it is a putative target gene for Oct-6, atranscription factor that is essential for timely peripheral myelination(Bermingham et al., Gene Develop 10:1751 (1996); Jaegle et al., Science273:507 (1996)). Second, its expression following nerve injury indicatesthat it encodes a myelin-related protein. Tramdorin, and/or its putativeligands, may provide avenues for the development of therapeutic agentsfor the treatment of the effects of demyclination or nerve injury, bothin the peripheral and central nervous systems. The ability to identifymolecules that promote myelination will significantly enhance theprospects for successfully using Schwann cells, and perhapsoligodendrocytes and olfactory ensheathing cells as well, to mitigatethe damage caused by MS and other demyelinating diseases. A significantuse of tramdorin will be in identifying such molecules.

VIII. Use of Tramdorin to Relieve Neurogenic and Neurogenic andNeuropathic Pain

[0258] Pain that arises from the nervous system is termed “neurogenic.”Peripheral neurogenic pain may follow transient pressure upon orstretching of a peripheral nerve or root, or reflect sustained damage toa nerve (“neuropathic pain”) such as in polyneuropathy, entrapmentneuropathy, or after herpes zoster. Neurogenic pain may have a centralorigin such as stroke, multiple sclerosis, or trauma, especially of thespinal cord.

[0259] Correct diagnosis allows tailoring treatment to thepathophysiologic mechanisms that trigger and maintain the painfulcondition. Assessing the pain location, intensity, quality, time course,precipitating and relieving factors, as well as its impact on physicaland psychosocial function is the first step in clinical analysis.Diagnosis depends upon, first, the neuroanatomical distribution of thepain and, second, evidence of sensory dysfunction involving a peripheralnerve, plexus, nerve root or central pathway. If the affected nerve orpathway is mixed motor and sensory, then weakness, muscle atrophy, orreflex abnormalities may be additional clues to neural involvement. Thediagnosis may be obvious but sometimes a thorough neurologicalexamination is needed to uncover the neurogenic origin of the pain. Apain drawing made by the patient frequently gives a good indication ofthe neuroanatomic distribution and quality of the pain.

[0260] Impaired sensation is often evident during a careful examination.Sensory dysfunction may be manifested as hypo- and/or hyperesthesia forone or more modalities, increasing pain to normally painful stimuli(hyperalgesia) or pain due to normally nonpainful stimuli (allodynia).Temporal and spatial sensory dysfunction are also common. Abnormalsensory function in neurogenic pain states can, regardless of modality,be described in terms of stimulus strength and sensation magnitude.Hypoesthesia consists of both increased perception threshold and reducedsensation magnitude at suprathreshold stimulus strengths. Occasionally,hypoesthesia occurs without an increase in stimulus detection threshold.Elevated stimulus perception threshold, together with an increase inslope of the magnitude/stimulus relation is typical for the combinationof hypo- and hyperesthesia often seen in neurogenic pain states. Evokedsensation hyperpathic syndrome often has a paresthetic or dysestheticcharacter or is frankly painful instead of the normal sensation evokedby the applied stimulus. Hyperesthesia can also occur separately with asteeper slope, and occasionally (thin line), with a lowered threshold.

[0261] Tramdorin1/mPAT2 has been shown to function as a proton-dependenttransporter of small amino acids. (See, Boll, M., Foltz, M.,Rubio-Aliaga, I., Kottra, G., and Daniel, H. (2002). FunctionalCharacterization of Two Novel Mammalian Electrogenic Proton-dependentAmino Acid Cotransporters. J Biol Chem 277, 22966-73.). It is related toyeast vacuolar and rat lysosomal amino acid transport proteins, butunlike tramdorin3/LYAAT-1, tramdorin1 does not appear to be a lysosomalprotein. Tramdorin1 lacks a putative lysosomal targeting motif (anacidic residue located in the C-terminus, −4 to −6 relative to adileucine, (LL(I,M,V); that is present in tramd3/LYAAT-1 consistent withits failure to co-localize with LAMp1. [see, Sandoval, I. V.,Martinez-Arca, S., Valdueza, J., Palacios, S., and Holman, G. D. . JBiol Chem 275, 39874-85. (2000).].

[0262] Tramdorin1/mPAT2 shows strong specificity for glycine, L-alanineand L-proline, with greatest inward currents generated by glycine.Although it is possible tramdorin1 could provide additional amino acidsrequired for metabolism in the paranodes and incisures, its restrictedsubstrate specificity and expression pattern suggest other function(s).Glycine can act as an inhibitory neurotransmitter in the CNS, and as anagonist of the NMDA class of ionotropic glutamate receptors. Glycinereceptors have not been reported in the PNS, but NMDA glutamatereceptors have been found on both adult Schwann cells and peripheralaxons (Coggeshall and Carlton, 1998; Fink et al., 1999; Kinkelin et al.,2000).

[0263] Therefore, in one embodiment, tramdorin1 could inhibitglutaminergic signaling in peripheral nerve by sequesteringextracellular glycine. Indeed it has been shown that axonal expressionof all three types of ionotropic glutamate receptor increases followingperipheral nerve inflammation; these receptors could contribute to thespontaneous discharges that have been observed in rat models of nerveinjury and inflammatory pain. Additionally, an increased frequency ofspontaneous discharges, with concomitant thermal hyperalgesia andmechanical allodynia, have been observed in saphenous nerves fromdysmyelinating prx mutant mice. See, Gillespie, C. S., Sherman, D. L.,Fleetwood-Walker, S. M., Cottrell, D. F., Tait, S., Garry, E. M.,Wallace, V. C., Ure, J., Griffiths, I. R., Smith, A., and Brophy, P. JPeripheral demyelination and neuropathic pain behavior inperiaxin-deficient mice. Neuron 26, 523-31 (2001).

[0264] These mice possess altered Schmidt-Lanterman incisures, andtherefore their expression of tramdorin1 may be reduced. In selectedembodiments, tramdorin1 may dampen neuronal excitability therebylimiting pain sensitivity. In other embodiments, tramdorin may modulateNMDA glutamate receptor signaling between Schwann cells, or betweenadjacent membranes of a single Schwann cell.

[0265] In one embodiment, it is contemplated that tramdorins may be to apatient suffering at least one symptom of neurogenic and/or neuropathicpain. In another embodiment, said administration is by direct injection.Once again, however, Applicants note that a the present invention is notlimited by tramdorins' precise mechanism of action or any specific modeof administration.

IX. Tramdorin Localization Using Antiserum

[0266] To characterize tramdorin1, an antiserum was raised against asequence near the N-terminus that was divergent among the tramdorin genefamily (e.g. antisera to a synthetic peptide: ESAKKLQSQDPSPANGTSC (SEQID NO: 74), containing amino acids 22-39 near the N-terminus oftramdorin1, as described in the section above.

[0267] In immunoblots of adult rat sciatic nerve, the antiserum binds toa single band close to the predicted molecular weight of unglycosylatedtramdorin1 (panel A of FIG. 29). A band of similar molecular weight isobserved on immunoblots of cells transfected with full-length mousetramdorin1 cDNA (panel B of FIG. 29). Transfected cells were stainedwith the anti-tramdorin1 antiserum, whereas parental cells wereunstained (panel C of FIG. 29). To localize tramdorin1, unfixedmyelinated fibers from rat sciatic nerves were labeled with thisantiserum. Tramdorin1 was localized in incisures and the paranodes, asshown by co-labeling with a monoclonal antibody against myelinassociated glycoprotein (MAG; panels A-C of FIG. 30). To determine iftramdorin1, like tramdorin3/LYAAT-1, is associated with lysosomes,teased fibers were double-labeled for tramdorin1 and LAMP1, a lysosomalmarker. Panels D-F of FIG. 30 shows that tramdorin1 and LAMP1 are foundin paranodes, but do not co-localize. While it is not intended that thepresent invention be limited to any particular motif of localization ora particular mechanism of action, this immunostaining suggest thattramdorin1 is localized to non-compact myelin, but is not a component oflysosomes.

[0268] Again, while it is not intended the present invention be limitedto any specific mechanism, Tramdorin1/mPAT2 has been shown to functionas a proton-dependent transporter of small amino acid. See, Boll, M.,Foltz, M., Rubio-Aliaga, I., Kottra, G., and Daniel, H. (2002).Functional Characterization of Two Novel Mammalian ElectrogenicProton-dependent Amino Acid Cotransporters. J Biol Chem 277, 22966-73.It is related to yeast vacuolar and rat lysosomal amino acid transportproteins, but unlike tramdorin3/LYAAT-1, tramdorin1 does not appear tobe a lysosomal protein. Tramdorin1 lacks a putative lysosomal targetingmotif (an acidic residue located in the C-terminus, -4 to -6 relative toa dileucine, (LL(I,M,V) that is present in tramd3/LYAAT-1. See,Sandoval, I. V., Martinez-Arca, S., Valdueza, J., Palacios, S., andHolman, G. D. (2000). Distinct reading of different structuraldeterminants modulates the dileucine-mediated transport steps of thelysosomal membrane protein LIMPII and the insulin-sensitive glucosetransporter GLUT4. J Biol Chem 275, 39874-85. Tramdorin1/mPAT2 showsstrong specificity for glycine, L-alanine and L-proline, with greatestinward currents generated by glycine (See, Boll supra). This restrictedsubstrate specificity and localization suggest that glycine and glycinetransport have a role in myelinating Schwann cells and modulatingphysiologies of the nervous system.

EXPERIMENTAL

[0269] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0270] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); ° C. (degrees Centigrade); RDA(representational difference analysis); nts (nucleotides).

Example 1

[0271] This example describes the identification of the cDNA for mousetramdorin 1. RDA on sciatic nerve from Oct-6 (−/−) and (+/+) sciaticnerves was performed as described (Bermingham et al., J Neurosci Res63:516 (2001); Erkman et al., Eur J Pharmacol 393:97 (2000)). The RDAprocedure was performed essentially as described elsewhere (Hubank etal., Nucleic Acids Res 22:5640 (1994); Lisitsyn et al., Science 259:946(1995); Adman et al., Biochem J 323:113 (1997)), with the followingmodifications: Carrier mRNA (Bermingham et al., manuscript inpreparation) was added to the total RNA for most experiments, prior tothe isolation of poly-A+ RNA and subsequent cDNA synthesis. cDNAs weredigested with DpnII, which cleaves at the recognition site GATC; becausethe four base 5′ overhang that results from its cleavage is identical tothat generated by BglII, the J, N, and R oligonucleotides that weredesigned by (Lisitsyn et al., Methods Enzymol 254:291 (1995)) for use ongenomic RDA with BglII were used here to amplify Dpn-II generatedfragments. The “N” adaptors were ligated to the DpnII ends. Thosefragments with two appropriately spaced adaptors were amplified by tworounds of PCR. Typically 50-600 bp fragments were amplified. A smallproportion of each of the drivers was digested with DpnII to remove the“N” adaptors, after which they were replaced with “R” adaptors. Each“tester” DNA was mixed with an excess of driver that was derived fromthe other source, denatured, and permitted to anneal. The reannealed DNAwas amplified by PCR, digested with mung bean nuclease (New EnglandBiolabs, Beverly, Mass.) to remove the single strands that result frompriming at only one end of a template, then amplified again by PCR. DNAwas digested with DpnII to remove the “R” adaptors, after which theywere replaced with “J” adaptors. This material was used as the testerfor a second round of subtraction, after mixing with an excess ofdriver. The products of the second round of subtraction were subjectedto two rounds of PCR amplification to produce the second differenceproducts, DP2. The linkers were removed from the DP2 DNA, after which itwas cloned. DpnII fragments were ligated into the BamHI site of pBKSH⁺(Stratagene, La Jolla, Calif.). Ligations were transfected into DH5α(Gibco-BRL, Grand Island, N.Y.).

[0272] Because Oct-6 is transiently expressed in sciatic nerve, theseexperiments were carried out on nerves that were isolated at P0 (day ofbirth), at or near the peak of Oct-6 expression. Six RDA cDNA clonescorresponded to genes that are downregulated in the absence of Oct-6,and therefore are putatively activated by Oct-6 in sciatic nerve(Bermingham et al., manuscript in preparation). The differentialabundance of these clones in cDNA from wild-type and Oct-6 mutantsciatic nerves was confirmed by hybridization to Southern blots ofPCR-amplified cDNA (“Snorthern blots”). These blots are reliableindicators of differential expression (Bermingham et al., J Neurosci Res63:516 (2001); Bermingham et al., manuscript in preparation). Asdescribed in Bermingham et al., J Neurosci Res 63:516 (2001), Snorthernblots use cDNA that was amplified by PCR for use as driver in the RDAexperiments described above. PCR reactions were optimized to ensure thatamplification was exponential, and parallel PCR reactions were pooled tominimize variation from individual samples and to obtain sufficientmaterial. Driver DNA (0.5 or 1 μg) was electrophoresed through 3%NuSieve 3:1 agarose (FMC) or 4% acrylamide gels, then transferred byconventional Southern blotting or electroblotting onto Hybond N⁺membranes (Amersham, Arlington Heights, Ill.).

[0273] The results for one clone, 125, is shown in FIG. 1A. RDA cloneJBSN125 contains a cDNA fragment that corresponds to a putative Oct-6regulated gene The panels show Southern blots of PCR-amplified cDNA(“Snorthern blots”) from Oct-6 (+/+) (wild-type) and Oct-6 (−/−)(mutant) sciatic nerves. The left-hand panel shows a Snorthern blot thathas been hybridized to RDA clone JBSN125; it indicates that one of twoinserts in this clone corresponds to a gene that requires Oct-6,directly or indirectly, for normal activation. The larger insert is notdifferentially expressed, and serves as an internal control. A subclonethat contains only the smaller insert, JBSN 125-10, also isdifferentially expressed (data not shown). The center and right panelsare control blots that have been hybridized with JBSN1 1, alacZ-containing clone, and Oct-6. No putative Oct-6 repressed genes wereisolated, except for LacZ and Neo, which are expressed in the knockoutmice. As expected, Oct-6 is expressed only in Oct-6 (+/+) sciaticnerves. The sizes of the fragments on the blots reflect the presence of24 bp linkers on the ends of the fragments. The differentially expressedsubclone, JBSN 125-10, was used to isolate additional sequence for thisgene.

[0274] Mouse cDNAs that correspond to clone 125-10 were isolated byobtaining and sequencing three homologous EST clones 1920302 (AI786604),1380757 (AI035402), and 1363993 (AI005767) (FIG. 1B). FIG. 1B showscDNAs that correspond to RDA clone JBSN125-10. Seven cDNA fragments,from two species (rat and mouse) are shown; they include the RDAfragment JBSN125-10, two independent, nonidentical 5′ RACE rat cDNAs, a3′ RACE rat cDNA, and three mouse EST cDNAs. Of these, the 2.45 kb mouseEST cDNA 1920302 (SEQ ID NO: 13) appears to include the entire openreading frame, which is depicted in black. The organism and tissue oforigin of each cDNA fragment is listed. The open reading frame commenceswith an ATG initiation codon that matches the Kozak consensus (Kozak,Nucleic Acids Res 15:8125 (1987)). EST clone 1380757 (SEQ ID NO: 50)contains a polyA tract just following the stop codon; because noconsensus polyadenylation signal exists in the cDNA near this site, thisclone probably represents an artifactual polyadenylation event. EST cDNAclones 1920302 (SEQ ID NO: 13) and 1363993 (SEQ ID NO: 43) diverge atthe 5′ splice site at the 3′ end of exon 7; comparison with genomicsequences indicates that EST cDNA 1363993 is unspliced. Theintron-derived reading frame contains only three amino acids before astop codon. Mouse EST 1363993 (AI005767) (SEQ ID NO: 43) thus appears tocorrespond to a partially spliced transcript.

[0275] The full-length mouse cDNA contains a 1.4 kb open reading frame(FIG. 2) that encodes a protein of 52 kd. The mouse cDNA and theputative protein that it encodes do not match any genes in the GenBanknon-redundant database, or the SwissProt database. Therefore, this cDNAis derived from a novel gene. FIG. 2 also shows a putative rat tramdorin1 cDNA, derived from 5′ and 3′ RACE sequences (see Example 2). FIG. 2shows the translated proteins; the mouse cDNA appears to encode a 478amino acid protein, while the corresponding rat protein is 481 aminoacids. Exon boundaries are superimposed on the mouse cDNA sequence. Theboundaries of exons 8, 9 and 10 are defined by comparison to Ensemb1mouse genomic sequence files, while the remainder are defined bysequencing of 129 mouse genomic DNA. The polyA tract of EST cDNA 1380757(SEQ ID NO: 50) commences at the A at position 1603. A putativepolyadenylation site is shown in bold; such AGUAAA (SEQ ID NO: 51)polyadenylation signals are processed in vitro at roughly 30% of theefficiency of the canonical AAUAAA (SEQ ID NO: 52) signal (Sheets etal., Nucleic Acids Res 18:5799 (1990)). The NlaIII restriction sites(CATG) that flank RDA clone 125-10 are shown in italics.

Example 2

[0276] This example shows the identification of a rat tramdorin 1 cDNA.Rat cDNAs that correspond to clone 125-10 were isolated by 5′ and 3′RACE (rapid isolation of cDNA ends (Frohman, Methods Enzymol 218:340(1993); Frohman, PCR Methods Appl 4:S40 (1994)). RNA was isolated fromsciatic nerves taken from one to two day old rat pups. First strand cDNAwas synthesized using Superscript II reverse transcriptase (Gibco-BRL).RACE was performed using a kit from Clontech, according to themanufacturer's instructions. The 3′ rat tramd1 RACE primer has thefollowing sequence:

[0277] 5′GACTTCCCCTGGCTGTGAAGAATGCGGGC 3′ (SEQ ID NO: 53)

[0278] and the 5′ rat tramd1 RACE primer has the following sequence:

[0279] 5′TcAGTCTGTGACAGAAGCGCTGGGCACATCtG 3′ (SEQ ID NO: 54)

[0280] The lower case nucleotides represent mismatches between the 5′RACE sequence and the final rat sequence. The final rat sequence has G'sreplacing the lower case letters in the 5′ primer.

[0281] The 5′ and 3′ rat RACE fragments combined define a 2.5kb cDNA(SEQ ID NO: 55). The full-length rat cDNA contains a 1.4 kb open readingframe (SEQ ID NO: 6) (FIG. 2) that encodes a protein of 52 kd (SEQ IDNO: 22). The rat cDNA and the protein that it encodes does not match anygenes in the GenBank non-redundant database, or the SwissProt database.Therefore, this cDNA is derived from a novel gene. The sequence of a rattramdorin1 cDNA, derived from 5′ and 3′ RACE sequences is shown in FIG.2 (SEQ ID NO: 6), as is the translated protein. The rat protein is 481amino acids.

Example 3

[0282] This example describes structural predictions for the proteinencoded by mouse tramdorin 1 and related tramdorins in humans.

[0283] The protein encoded by mouse cDNA 1920302 (SEQ ID NO: 13) waspredicted by several protein structure prediction programs to encode atransmembrane protein. These programs use aspects of protein structuresuch as hydropathy and charge distributions and/or utilize differentalgorithms to arrive at their predictions. Although all the programsused predicted the protein would be a polytopic transmembrane protein,they differed in the number of transmembrane domains predicted. PHDHtm(Rost et al., Protein Sci 4:521 (1995)) and TM finder (Deber et al.,Protein Sci 10:212 (2001)) predicted 8 transmembrane domains (FIG. 3Aand data not shown). PRED-TMR (Pasquier et al., Protein Eng 12:381(1999)) predicted the presence of 10 putative transmembrane domains, andan Argos hydropathy plot (Argos et al., Eur J Biochem 128:565 (1982))predicted 10 hydrophobic domains with a possible 11^(th) (data notshown). Memsat2 (PSIPRED; McGuffin et al., Bioinformatics 16:404(2000)), and TMHMM (Sonnhammer et al., Proc Int Conf Intell Syst MolBiol 6:175 (1998)) predict that the protein contains 11 transmembranehelices with the N-terminus facing the cytoplasm (FIG. 3). Regardless ofthe differing predictions about the number of transmembrane domains, ortheir topology, this protein appears to contain multiple transmembranehelices. Therefore we have named the putative gene product tramdorin,for transmembrane domain rich protein. Tramdorin has been assigned thehuman and mouse gene symbol tramd1 .

[0284] The putative amino acid sequence encoded by the putative fulllength EST cDNA 1920302 (SEQ ID NO: 13) is shown in FIG. 3A. The aminoacid sequence of EST cDNA 1920302 (SEQ ID NO: 14) was analyzed using thetransmembrane domain prediction programs PHDHtm (Rost et al., ProteinSci 4:521 (1995)), Memsat2 (McGuffin et al., Bioinformatics 16:404(2000)), and TMHMM (Sonnhammer et al., Proc Int Conf Intell Syst MolBiol 6:175 (1998)). 11 putative transmembrane domains are numbered.Transmembrane domains predicted by PHDHtm and TMHMM are marked withCapital T's. For Memsat2, transmembrane domains are marked as follows:O: outside transmembrane helix cap; X: central transmembrane helixsegment; I: inside transmembrane helix cap. For both Memsat2 and TMHMM,predicted cytoplasmic domains are marked by “+”, while non-cytoplasmicdomains are marked by “−”. The partially spliced EST cDNA 1363993 (SEQID NO: 43) is truncated following putative transmembrane domain #6.Three consensus glycosylation sites within the protein sequence areshown in bold. FIG. 3B shows a diagram depicting the predicted topologyof tramdorin1 with 11 transmembrane domains, as predicted by Memsat2 andTMHMM. The three extracellular/lumenal glycosylation sites are markedwith branched structures. While it is not intended that the presentinvention be limited to any particular level of gylcosylation (if any)the electrophoretic mobility of tramdorin suggests that it is notglycosylated extensively. Moreover, glycosylation is shown in black atonly one site, in accordance with the observation that in transmembranedomain proteins with multiple glycosylation sites, only one is typicallyused (Landolt-Marticorena et aL, Biochem J 302:253 (1994)); the othersare shown in gray.

[0285] While a precise understanding of the structure of tramdorin isnot required for the practice of the present invention, the followingconsiderations suggest that tramd proteins consist of 11 transmembranedomains, with the N-terminus facing the cytoplasm. However, theinvention is not to be limited to such a structural prediction oftramdorin proteins. First, those programs that predicted 11transmembrane domain helices more accurately predicted the number andtopology of the transmembrane helices of a set of transmembrane proteinsof known structure (Tusnady et al., J Chem Inf Comput Sci 41:364 2001).Therefore those programs also may be better at predicting the structureof unknown proteins. Although PHDHtm, which predicted 8 transmembranedomains, has been shown to be up to 90% accurate in predictingtransmembrane helices, the predicted helices often were too long.Consistent with this observation, the first, third and seventhtransmembrane domains predicted by PHDHtm are much longer than typicaltransmembrane domains (23-30 amino acids; reviewed in (von Heijne, ProgBiophys Mol Biol 66:113 (1996)), and are predicted to be twotransmembrane domains by memsat2 and TMHMM. Second, analysis of humantramd1 , -2, and -3 using Memsat2 and TMHMM produced 11 transmembranepredictions similar to those shown for mouse tramd1 in FIG. 3, exceptthat TMHMM did not predict the first transmembrane domain for humantramdorin 1. Third, if the protein were to possess 10 transmembranedomains, analysis of charged amino acids flanking the first putativetransmembrane domain (as predicted by either PHDHtm or PRED-TMR)suggests that the N-terminus will be outside the cytoplasm (Hartmann etal., PNSA USA 86:5786 (1989)). Such a topology could arise from cleavageof an N-terminal signal sequence. However, a signal sequence predictionprogram (Nielsen et al., Protein Eng 10:1 (1997)) indicates that theprotein does not appear to contain a cleaved N-terminal signal sequence.Fourth, membrane proteins are glycosylated only on their extracytoplamicfaces, and the distribution of glycosylation consensus sites(NX^(S)/_(T), [Asn Xaa Ser/Thr] (SEQ ID NO: 56) wherein X [Xaa] is anyamino acid except proline) supports the Memsat2 and TMHMM topology. Themouse tramd1 protein sequence contains five putative glycosylationsites. Of these, an N-terminal NGT [Asn Gly Thr] sequence is notconserved in human, and an NIS [Asn Ile Ser] sequence is buried inpredicted transmembrane helix 5. The three conserved, non-transmembranedomain consensus glycosylation sites are predicted to beextracytoplasmic in the structures predicted by Memsat2 and TMHMM. Thusthe preponderance of evidence from protein structure prediction programssuggests that the protein contains 11 transmembrane domains, with theN-terminus facing the cytoplasm (FIG. 3B).

[0286] However, as noted above, the invention is not limited to anyparticular tramdorin structure, and several considerations suggestalternative structures for tramdorins. The 11 transmembrane model fortramdorin structure. Proteins with 11 transmembrane domains are veryrare (Jones, FEBS Lett 423:281 (1998)). Furthermore, the closest knownrelatives of tramdorins, the vesicular GABA transporters, are thought toconsist of 10 transmembrane domains; they correspond to predictedtransmembrane domains 2-11 of tramdorin1. For the C. elegans vesicularGABA transporter AF03 1935.1, the memsat2 program predicted 10transmembrane domains, but the orientation in the membrane was oppositethat shown in (McIntire et al., Nature 389:870 (1997)), while TMHMMpredicted 9 transmembrane domains (data not shown). These observationsraise the possibility that current transmembrane domain structureprediction programs may have difficulty predicting correctly thestructure of vesicular GABA transporters, and may have difficulty withtramdorins as well. Thus, the theoretical prediction that tramdorinsconsist of 11 transmembrane domain proteins with cytoplasmic N-terminishould not be considered limiting for the purposes of the presentinvention cDNAs that encode truncated tramdorin proteins have beenidentified for both human and mouse tramd1, and for tramd3. In the caseof mouse and human tramd1, the cDNAs that encode truncated proteinsarise from the failure to splice out the intron that follows exon 7.This intron is unusual in that it possesses 3 regions of homologybetween mouse and human, clustered at its 5′ end (FIG. 18). While not tobe limited to any particular mechanism, if these conserved regions ofhomology are related to the splicing of exon 7, they may indicate abiological role for the truncated proteins. Such proteins would containthe first six putative transmembrane domains.

Example 4

[0287] This example is directed to the expression pattern of tramdorin 1in the mouse. To determine the size of tramdorin mRNA(s) and todetermine if tramdorin expression is restricted to sciatic nerve, anorthern blot with RNAs from adult mouse sciatic nerve and other tissueswas hybridized to a radiolabelled probe synthesized from tramdorin cDNA1920302 (SEQ ID NO: 13) (See, FIG. 4). Specifically, Each lane containsan equal amount (10 μg) of total RNA isolated from the indicated mousetissues. The blot was successively hybridized to a radiolabeled probecorresponding to tramdorin1, and exposed to film for 14 days after thehybridization. The sizes of the transcripts are estimated according tothe sizes of 28S and 18S rRNAs (4712 nt and 1869 nt, respectively;(Hassouna et al., 1984; Raynal et al., 1984).

[0288] The probe hybridized strongly to two distinct transcript classesin sciatic nerve, of approximately 2 kb and 2.8 kb in size. Of these,hybridization was strongest to the 2 kb transcripts. The sizes of thetranscripts were based on the sizes of 28S and 18S rRNAs (4712 nt and1869 nt, respectively; (Hassouna et al., Nucleic Acids Res 12:3563(1984); Raynal et al., FEBS Lett 167:263 (1984)). No expression wasobserved in spleen, and only a very faint band was seen at >6.5 kb incerebrum, indicating that tramdorin 1 is not ubiquitously expressed. The2.8 kb species is the most prominent tramdorin 1 message in lung,adrenal gland and thymus. This result indicates that EST cDNA 1920302(SEQ ID NO: 13) defines a nearly full length tramd1 message, and mustcorrespond to the larger transcript class, because it is approximately2.45 kb, excluding the polyA tail. Therefore tramdorin1 expression isneither ubiquitous, nor restricted to sciatic nerve. In contrast, asimilar northern using rat tissue did not show hybridization to lung andthymus (data not shown), suggesting either variation in tramdorin1expression among species, or cross-hybridization to related sequences.

[0289] Whether the expression of tramd1 in sciatic nerve is modulated bynerve injury was of interest. If so, its pattern of expression woulddetermine if the genes are expressed in myelinating ornon-myelinating/immature Schwann cells. Axotomy leads to a sharp declinein the steady state mRNA levels for myelin-related genes, and anincrease in the expression of genes associated with immature Schwanncells. Subsequently, in crushed nerves, Schwann cells re-expressmyelin-related genes in response to contact with regenerating axons(reviewed in Poduslo, “Regulation of myelin gene expression in theperipheral nervous system.” In Dyck, P. J. et al. (eds), PeripheralNeuropathy, W. B. Saunders Co., Philadelphia, pp. 282-289 (1993)).

[0290] A northern blot of RNA from sciatic nerves collected at varioustimes following crush or transection injury was hybridized with a mousetramd1 probe (FIG. 5). It should be noted that the mouse tramd1 probealso hybridized with a northern blot of RNA from rat nerve tissue. AdultSprague Dawley rats were anesthetized with 50 mg/kg pentobarbitol i.p.,and the sciatic nerves were exposed at the obturator tendon. To preventaxonal regeneration, nerves were doubly ligated and transected betweenthe ligatures. Nerves were crushed by compression with flattened forcepstwice, each time for 10 seconds. Animals were allowed to survive forvarious periods of time prior to sacrifice by CO₂ inhalation. For RNAextraction, several millimeters of nerve adjacent to the lesion sitewere trimmed off, and the distal nerve-stumps were frozen in liquidnitrogen. Where indicated, the distal stumps of crushed nerves weresubdivided into proximal and distal segments of equal lengths.Unlesioned nerves were taken from animals of various ages. Total RNA wasisolated from the sciatic nerves by CsCl₂ gradient centrifugation(Chirgwin et al., Biochem 18:5294 (1979)). Equal samples (10 μg) of RNAwere electrophoresed in 1% agarose, 2.2 M formaldehyde gels, transferredto nylon membranes in 6×SSC, and UV cross-linked (0.12 joules). Blotswere prehybridized, hybridized, and washed using standard techniques(Sambrook et al., Molecular Cloning: a Laboratory manual. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. [1989] pp. 31). cDNA probeswere ³²P-labeled with specific activities of 2-5×10⁹ cpm/μg, prepared byprimer extension with random hexamers using the Prim-a-gene kit(Promega) according to the manufacturer's instructions.

[0291] Northern blots of RNA from segments of sciatic nerve distal to atransection injury (in which axons do not regenerate) and crush injury(in which axons regenerate and are remyelinated) were hybridized withprobes for tramdorin, and Oct-6. The blots were hybridized also toprobes for P0, p75 nerve growth factor receptor (NGFR) and GAPDH ascontrols. For the crushed nerves, RNA was made from two sciatic nervesegments, one immediately distal to the injury (P) and one more distalto the injury (D) (FIG. 5). Myelination occurs more slowly in the (D)segments because contact with regenerating axons is delayed.

[0292] Tramd1 expression is downregulated by transection injury, andremains low. Following a crush injury, tramd1 is downregulated, butsubsequently it is reexpressed as axons regenerate and are remyclinated(FIG. 5). Therefore, the tramdorin1 gene encodes a myelin-relatedprotein. Several genes that are required for normal myelination showthis pattern of expression; these include Oct-6, P0, connexin32 andPMP-22. (Poduslo, “Regulation of myclin gene expression in theperipheral nervous system.” In Dyck, P. J. et al. (eds), PeripheralNeuropathy, W. B. Saunders Co., Philadelphia, pp. 282-289 (1993);Scherer et al., J Neurosci 14:1930 (1994); Sohl et al., Eur J Cell Biol69:267 (1996)). This result suggests that tramd1 may play an importantrole in the timely formation of peripheral myelin.

Example 5

[0293] This example describes experiments to determine the mousechromosomal location of tramd1.

[0294] The mouse chromosomal location of Tramd1 was determined byinterspecific backcross analysis using progeny derived from matings of[(C57BL/6J x Mus spretus)F₁ X C57BL/6J] mice. This interspecificbackcross mapping panel has been typed for over 3100 loci that are welldistributed among all the autosomes as well as the X chromosome(Copeland et al., Trends Genet 7:113 (1991)). C57BL/6J and M spretusDNAs were digested with several enzymes and analyzed by Southern blothybridization for informative restriction fragment length polymorphisms(RFLPs) using a mouse cDNA probe specific for Tramd1. The 7.5 kb BglI M.spretus RFLP (see below) was used to follow the segregation of theTramd1 locus in backcross mice. The mapping results indicated thatTramd1 is located in the proximal region of mouse chromosome 11 linkedto Il13 and Hand1.

[0295]FIG. 6A shows a summary of the mapping results. Each columnrepresents the chromosome identified in the backcross progeny that wasinherited from the (C57BL/6J x M. spretus) F₁ parent. The shaded boxesrepresent the presence of a C57BL/6J allele and white boxes representthe presence of a M. spretus allele. The number of offspring inheritingeach type of chromosome is listed at the bottom of each column. Apartial chromosome 11 linkage map showing the location of Tramd1 inrelation to linked genes is shown in FIG. 6B. Recombination distancesbetween loci in centimorgans are shown to the left of the chromosome andthe positions of loci in human chromosomes, where known, are shown tothe right. References for the human map positions of loci cited in thisstudy can be obtained from GDB (Genome Data Base), a computerizeddatabase of human linkage information maintained by The William H. WelchMedical Library of The Johns Hopkins University (Baltimore, Md.).

[0296] Although 136 mice were analyzed for every marker and are shown inthe segregation analysis (FIG. 6A), up to 179 mice were typed for somepairs of markers. Each locus was analyzed in pairwise combinations forrecombination frequencies using the additional data. The ratios of thetotal number of mice exhibiting recombinant chromosomes to the totalnumber of mice analyzed for each pair of loci and the most likely geneorder are: centromere-Il13-1/142-Tramd1-6/179-Hand1. The recombinationfrequencies (expressed as genetic distances in centiMorgans (cM)±thestandard error) are-Il13-0.7 +/−0.7-Tramd1-3.4 +/−1.4-Hand1. Thisinterspecific map of chromosome 11 has been compared with a compositemouse linkage map that reports the map location of many uncloned mousemutations (provided from Mouse Genome Database, a computerized databasemaintained at The Jackson Laboratory, Bar Harbor, Me.). Tramd1 mapped ina region of the composite map that lacks mouse mutations with aphenotype that might be expected for an alteration in this locus (datanot shown).

[0297] Interspecific backcross progeny were generated by mating(C57BL/6J x M spretus)F₁ females and C57BL/6J males as described(Copeland et al., Trends Genet 7:113 (1991)). A total of 205 N₂ micewere used to map the Tramd1 locus, as described above. DNA isolation,restriction enzyme digestion, agarose gel electrophoresis, Southern blottransfer and hybridization were performed essentially as described(Jenkins et al., J Virol 43:26 (1982)). All blots were prepared withHybond-N⁺ nylon membrane (Amersham). The probe, a 503 bp BssHII/EcoRIfragment from the 3′ UTR of cDNA 1920302, was labeled with [α³²P] dCTPusing a random primed labeling kit (Stratagene); washing was done to afinal stringency of 0.5×SSCP, 0.1% SDS, 65° C. A fragment of 10.0 kb wasdetected in BglI digested C57BL/6J DNA and a fragment of 7.5 kb wasdetected in BglI digested M. spretus DNA. The presence or absence of the7.5 kb BglI M. spretus-specific fragment was followed in backcross mice.A description of the probes and RFLPs for the loci linked to Tramd1including Il13 and Hand1 has been reported previously (Cross et al.,Development 121:2513 (1995); McKenzie et al., J Immunol 150:5436(1993)). Recombination distances were calculated using Map Manager,version 2.6.5. Gene order was determined by minimizing the number ofrecombination events required to explain the allele distributionpatterns.

[0298] The proximal region of mouse chromosome 11 in which tramd1 maps,is a region that is syntenic with human chromosome 5q. These resultsindicate that tramd1 is located on human 5q31-33, suggesting that thehuman homolog of Tramd1 will map to 5q31-33 as well. An autosomalrecessive Charcot-Marie-Tooth Syndrome demyelinating neuropathy has beenmapped to this region (Guilbot et al., Ann NY Acad Sci 883:56 (1999a);Guilbot et al., Eur J Hum Genet 7:849 (1999b); LeGuern et al., Hum MolGenet 5:1685 (1996)), suggesting that tramdorin1 could be a candidateperipheral neuropathy disease gene.

Example 6

[0299] This example describes experiments directed at studying the humantramdorin gene. As a first step toward determining if tramdorin1mutations are associated with human peripheral neuropathy, the humantramdorin gene was studied. Six human EST cDNAs that possess homology tomouse tramdorin1, I.M.A.G.E numbers 3184556 (BE501426), 1738130(AI140615), 1837427 (AI208756), 1388139 (AA843982), and 2549054(AI953890), as well as EST cDNA DKFZp434G1123 from the DeutschesRessourcezentrum für Genomforschung GmbH (RZPD) were obtained andsequenced in their entirety. The sequences were aligned on human genomicDNA from the International Human Genome Sequencing Consortium (IHGSC)Homo sapiens chromosome 5 working draft sequence segment NT_(—)006951.4and Celera databases (Lander et al., Nature 409:860 (2001); Venter etal., Science 291:1304 (2001)). These cDNAs showed greatest homology tofour discrete locations within 250 kb of chromosome 5q sequence (FIG.7), confirming the mapping of tramdorin in mouse, but in addition,suggesting the existence of multiple tramdorin genes. These genes havebeen assigned the names tramd1, tramd2, tramd3, and tramdL.

[0300] In FIG. 7, the IHGSC sequences are shown; coordinates indicatekilobases of nucleotide sequence. The coordinates correspond to scaffoldNT_(—)006951.4 (1038-1290 kb). Human genomic sequences that are mosthomologous to mouse tramdorin 1 EST 1920302 reside between 70 and 102kb. Therefore this region, and EST cDNA 3184556, are likely to representthe human homolog of mouse tramd1.

[0301] The human tramd1 gene has greatest homology to the original mousetramd1 gene, and is flanked on its 3′ end by tramd2, and on its 5′ endby tramd3, which is transcribed in the opposite orientation to tramd1and tramd2. Nested between tramd1 and tramd3 resides a tramdorin genefragment, tramdL, in the same orientation as tramd1 and tramd2. Itconsists only of 3′ tramdorin sequences, and is defined by a single ESTcDNA, 1388139. All four putative genes are found on both the Celera andIHGSC sequences. The structures of individual tramdorin genes are shownin greater detail as described below.

[0302]FIG. 8 presents the organization of the human tramdorin loci.Sequences are from Celera human chromosome 5 scaffold sequence GAx2HTBL3TT27:2500000-3000000, except where specified. The human tramdorin1 locus is shown in FIG. 8A. For cDNA htramd1 #1, the dashed linerepresents an apparent non-splicing event has joined sequences that arelocated 15.7 kb apart in the genome. The human tramdorin 2 locus isdepicted in FIG. 8B. Exons 1-7 were identified by homology to mousetramd1 and/or human tramd3. Exons 8-10 were identified by homology toEST cDNA 1837427. The human tramdorin 3 locus is depicted in FIG. 8C.Sequence from Celera, except 1180 nt of ambiguous sequences betweenexons 3 and 4 were replaced with corresponding sequence from IHGSCsequence NT_(—)006951.3, as represented by the break marks between exons3 and 4. Three EST cDNAs contain sequences that are homologous to mousetramd1 ; a fourth, 1845574, contains sequence that are homologous to the3′ end of EST cDNA 1738130. These cDNAs reveal that expression of thetramd3 gene generates multiple alternative transcripts. EST cDNA 1388139contains sequences that correspond to both chromosome 5q and to theSTK4/Mst-1/Krs-2 locus on chromosome 20 (FIG. 8D); the correspondinggenomic regions are diagrammed. This EST encodes only one exon that ishomologous to mouse tramdorin 1; on the chromosome 5q genomic sequence,no other exons with homology to mouse or human tramdorin genes was foundwithin 20 kb of this exon. This locus does not encode a completetramdorin protein, and therefore is called tramdorin L.

[0303]FIG. 9 shows sequence between nucleotides 50001 and 300000 of Homosapiens chromosome 5 working draft sequence segment NT_(—)006951.4. Eachline of the figure has 100 nucleotides and each page has 7000nucleotides. At the top of each page, the coordinates on sequenceNT_(—)006951.4 of the first nucleotide on that page is given. Knownexons for tramdorin 1, tramdorin 2, tramdorin 3 and tramdL are shown inbold type and are also indicated in the right margin. These exons wereidentified by their inclusion in human cDNAs or are inferred by homologyto the homologous mouse gene, or to paralogous human sequences.Initiation ATG and stop codons, and putative polyadenylation signalsites are shown in bold italic. Note that tramd3 is in the oppositeorientation relative to the other tramd genes, and is shown in theantisense orientation. Note that a single nucleotide polymorphism (C-T)in exon 2 of human tramdorin 1 at position 222682 has been confirmed,resulting in a synonymous change of a leucine codon. Other polymorphismsmay also exist.

[0304] The structure of the human tramdorin 1 gene was determined byidentifying regions of homology between the mouse and human tramdoringenes using the Pustell homology program (Pustell et al., Nucleic AcidsRes 10:4765 (1982b)). This program identified both putative exons (FIGS.8A and 9) as well as other conserved sequences that may definetranscriptional or splicing enhancers. Additional putative transcribedregions of the tramdorin 1 gene were defined by the EST cDNA 3184556 anda cDNA that was isolated from a human sciatic nerve cDNA library kindlyprovided by Drs. James Lupski and Cornelius Berkoel of BaylorUniversity.

[0305] Exons 1-10 of the tramdorin 2 gene are depicted in FIGS. 8B and9; they are defined by a single EST cDNA, 1837427, and by homology withtramdorins 1 and 3, and mouse tramdorin 2 cDNA. It should be noted thatmouse tramd2 cDNA was used to identify the first exon. The known andinferred exons span 26 kb of genomic DNA.

[0306] The sequences of 4 alternatively spliced tramdorin 3 EST cDNAswere used to infer the structure of the tramdorin 3 gene (FIGS. 8C and9). These cDNAs possess distinct sets of exons that together span 50 kbof genomic DNA. They demonstrate that expression of the tramd3 involvesextensive alternative RNA processing to generate transcripts that encodetramd3 proteins with 3 distinct C termini. In addition, the distinct 5′ends of EST cDNAs 2549054 and 1738130 could indicate the presence of twotramd3 promoters.

[0307] A fourth tramdorin-related sequence, tramdL, is defined by theEST cDNA 1388139 (SEQ ID NO: 23)(FIGS. 8D, 9 and 25). tramdL containsonly sequences that are homologous to exon 10 of tramdorin1. This cDNAis derived from a parathyroid adenoma, and is unusual in that its 5′ endconsists of sequences derived from a member of the STE20 family ofkinases, STK4/Mst-1/Krs-2 (FIG. 12A; Creasy et al., J Biol Chem270:21695 (1995); Taylor et al., PNSA USA 93:10099 (1996)), located onhuman chromosome 20. The STK4/Mst-1/Krs-2 cDNA fragment is fused to 3′sequences of tramdL (FIG. 12B), located on chromosome 5. HumanSTK4/Mst-1/Krs-2 and tramdL sequences. EST cDNA 1388139 encodes a fusiongene product that fuses 5′ MST kinase sequences to tramdorin 3′sequences at a splice junction. Genomic sequence from nucleotides8189548 to 8196472 of human chromosome 20 contig NT011382, which containexons 6-9 of the serine-threonine kinase STK4/Mst-1/Krs2, are shown inFIG. 12A. The 5′ end of EST cDNA 1388139, within exon 6, is shown as anasterisk in the genomic sequence. The portion of the conserved kinasedomain that is encoded by exons 6-8 is shown in bold. The caspase-3cleavage site that activates the kinase is shown in bold italic. In ESTcDNA 1833139, exon 9 is spliced to tramdL on human chromosome 5. Thenucleotide sequence of tramdL EST cDNA 1388139 (SEQ ID NO: 23) is shownin FIG. 25. Vector sequences 5′ to the EcoRi site (GAATTC) and 3′ to thepolyA stretch have been deleted, as have all but 11 A's in the polyAstretch. The point at which the cDNA diverges at its 5′ end fromSTK4/Mst-1/Krs2 sequences is marked with a dot (). Sequences betweenthis point and the termination codon are translated (SEQ ID NO: 71).MscI (TGGCCA) and DraI (TTTAAA) sites that were used to generate atramdL probe are underlined. Human chromosome 5 genomic sequence betweennts 55336 and 56128 on Celera scaffold sequence GAx2HTBL3TT27:2500000-3000000 is shown in FIG. 12B, along with an openreading frame that is significantly conserved with the C-tennini oftramd 1-3. An asterisk following the open reading frame denotes atermination codon, whereas the asterisk in the sequence denotes thelocation of the polyadenylation site of EST cDNA 1833139 (FIG. 12B). Thefusion between STK4/Mst-1/Krs-2 and tramd L involves the 5′ splice siteof STK4/Mst-1/Krs-2 exon 9, and a 3′ splice site of tramdorin L (FIG.12C). Such a fusion is unlikely to result from an artifact of cDNAsynthesis. A comparison of proteins encoded by STK4/Mst-1/Krs-2 and ESTcDNA 1833139 is shown in FIG. 12D. Caspase-3 cleaves the peptidesequence DEMD (SEQ ID NO: 57), thereby separating active p36 kinase fromthe 3′ inhibitory domain. In the putative STK4/Mst-1/Krs2-TramdL fusionprotein (SEQ ID NO: 71), the C-terminus of STK4/Mst-1/Krs2 is replacedby tramdL peptide sequences that include a complete transmembranedomain, shown inserted in a membrane. The kinase activity of C-terminaldeletions of STK4/Mst-1/Krs-2 (Creasy et al., J Biol Chem 271:21049(1996)) suggests that an STK4/Mst-1/Krs-2-tramdL fusion protein wouldretain some kinase activity.

[0308] Comparison of the genomic sequences for STK4/Mst-1/Krs-2 (SEQ IDNO: 27) and tramdL (SEQ ID NO: 29) reveal that the fusion sitecorresponds to a 5′ splice site in the STK4/Mst-1/Krs-2 gene, and a 3′splice site in tramdL (FIG. 12C). Such a fusion is unlikely to be anartifact of cDNA synthesis. This observation raises the possibility thattramdL resides near a parathyroid tumor translocation breakpoint,between chromosomes 20 and 5, that results in expression of aSTK4/Mst-1/Krs-2-tramdorinL fusion protein. The predicted fusion proteinencoded by EST cDNA 1388139 is depicted in FIG. 25 (SEQ ID NO: 71), anda predicted fusion protein sequence containing additional 5′ human STK4sequence is depicted in FIG. 26 (SEQ ID NO: 44). STK4/Mst-1/Krs-2contains a C-terminal inhibitory domain (Creasy et al., 1996) that iscleaved by Caspase-3 to generate a cleavage product, p36, with kinaseactivity (Graves et al., Embo J 17:2224 (1998); Lee et al., Oncogene16:3029 (1998); Watabe et al., J Biol Chem 275:8766 (2000)). TheSTK4/Mst-1/Krs-2-tramdorinL fusion protein would replaceSTK4/Mst-1/Krs-2 C-terminal sequences with a C-terminal transmembranedomain (FIG. 12D). Based on the activity of C-terminal deletions ofSTK4/Mst-1/Krs-2 (Creasy et al., J Biol Chem 271:21049 (1996)), thefusion protein may retain kinase activity, and may represent animportant aspect of the etiology of parathyroid tumors. However, thepresent invention does not require an understanding of the underlyingmechanism, and the invention is not to be limited to any particularmechanism.

Example 7

[0309] This example describes the expression pattern of human tramdoringenes. To determine the tissues in which the various tramdorin genes areexpressed, a northern blot of RNA derived from 12 human tissues wassequentially hybridized to radiolabelled cDNA probes that correspond totramd1, 2, 3 and tramdL (FIG. 13). A human multitissue northern blot waspurchased from OriGene Technologies, Rockville, Md., and probedsequentially with a 440 bp EcoRI+Nco fragment of human tramd2EST1837427, a 340 bp MscI-DraI fragment of human tramdL EST1388139, a850 bp StuI+KpnI fragment of human tramd3 EST2549054, a 355 bpEcoRI+PvuII fragment of human tramd1 EST3184556, and GAPDH.Hybridization to a GAPDH probe shows that RNA is present in all lanes.Phosphorimager exposure times were 2 weeks for tramdorins 1 and 2, oneweek for tramd3 and overnight for GAPDH.

[0310] Each probe produced a distinct hybridization result (see FIG.13). The tramd1 probe hybridized to a 4 kb transcript that appears to beexpressed only in muscle and kidney. Tramd2 appears to be encoded by a 3kb transcript that is expressed only in testes. Tramd3 is the mostwidely expressed of the tramd genes; it is encoded by a 7 kb transcriptthat is present in every tissue except liver and muscle. It is mosthighly expressed in brain, in which an alternative 9 kb tramd3transcript is seen also. Its pattern of expression is nearlycomplementary to that of tramd1 ; both genes are expressed together onlyin kidney. In contrast to tramd1-3, no expression was observed fortramdL (data not shown). Either the gene is not expressed, consistentwith the inability to find 5′ sequences for it, or it is expressed onlyin tissues not represented on the northern blot. The differences in thesizes of transcripts that are hybridized by the various tramd probesalso demonstrates that in spite of the considerable amino acidconservation between the tramd proteins, cross-hybridization between thetramd genes in minimal under the conditions used.

Example 8

[0311] This example demonstrates the conservation among humantramdorins. Translation of the known putative coding exons fortramdorins 1 , 2 and 3 indicate that these proteins are highly conserved(FIG. 14). Comparison was by the Clustal W algorithm (Thompson et al.,Nucleic Acids Res 22:4673 (1994)). Tramd1, tramd 2 and tramd3 appear tohave ten coding exons except for the N-terminus (first coding exonsequences). Consensus glycosylation sites in the putative secondextracytoplasmic loop (FIG. 3), amino acids 181-183 and 190-192 areconserved in tramdorins 1 and 3, but not in tramdorin 2.

[0312] Example 9

[0313] This example demonstrates the conservation of tramdorin proteinsbetween different species.

[0314] Clustal W analysis of putative tramdorin 1 proteins from human,rat, mouse and two invertebrate proteins that were identified by genomesequencing is shown in FIG. 15. The most closely related sequences totramdorins are putative proteins that have been derived from theDrosophila, Caenorhabditis elegans and Saccaromyces cerevisiae genomesequences (FIG. 15). Mouse tramd1 protein is 39% identical to Drosophilaprotein CG13384, and 32% identical to the T27A1.5 protein from C.elegans. The functions of these proteins in invertebrates may be relatedto tramdorin function in vertebrates. If so, tramdorins would berequired for a function that is conserved by evolution, and predates theorigin of myelin. The vertebrate proteins to which the tramdorins aremost closely related are vesicular γ-aminobutyric acid (GABA)transporters (VGATs; FIG. 16; (McIntire et al., Nature 389:870 1997;Sagne et al., FEBS Lett 417:177 (1997)), to which mouse tramd1 is 21-22%identical. Note that transmembrane domains 1-10 of (McIntire et al.,Nature 389:870 (1997)) correspond to predicted transmembrane domains2-11 of tramd1. GABA is the principal inhibitory neurotransmitter in thebrain (reviewed in Zigmond, Fundamental Neuroscience, Academic Press,San Diego Vol., pp.xvi, 1600 (1999)), and VGATs are thought to pack GABAinto vesicles prior to exocytosis into the synapse. The similaritybetween VGATs and tramdorins suggests that the latter may also transportsmall molecule(s) with significant biological importance. Furthermore,the observation of neurons with GABA, but without VGAT, suggests theexistence of additional GABA transporters (Chaudhry et al., J Neurosci18:9733 (1998)).

Example 10

[0315] This example presents the structure of the mouse tramdorin 1 andtramdorin 3 genes.

[0316] The organization of the mouse tramd1 gene was studied tounderstand better the origin of the alternatively spliced tramdorincDNAs, and as a preliminary step toward generating mice that lack tramd1function. A 129 mouse genomic library in bacteriophage λ was screenedwith a probe consisting of a 720 bp XhoI-NcoI fragment from cDNA 1920302that contains sequence from exons 1-6. Three phage were isolated (FIG.17A). In addition, a 129/Sv bacterial artificial chromosome (BAC)library (Research Genetics, Huntsville, Ala.) was screened using thepolymerase chain reaction (PCR) with primers 5′ GCT GCC ACA AGA ACG AGACG 3′ (SEQ ID NO: 58) and 5′ ATG ATG ACC AGG CTG ACC AGC 3′ (SEQ ID NO:59), that amplify 169 bp of exon 6. A single tramdorin-containing BACwas isolated. Subclones from the BAC and from the lambda phage weresequenced to determine the structure of the 5′ end of the tramdoringene. A partial restriction map of mouse tramd1 is shown at the top ofthe FIG. 17A. The exact location of exon 5 relative to exons 4 and 6 isunknown, and the sizes of introns between exons 7-10 are unknown. Shownbelow the map are three tramd1 EST cDNAs that define tramd1 exons.Genomic regions that have been sequenced are shown as thick lines belowthe cDNAs, and below them, select subcloned fragments are shown (FIG.17A). The genomic sequence 3′ to exon 7 corresponds to sequence in ESTcDNA 1363993, indicating that this cDNA is partially spliced. Exons 8,9,and 10 are defined by sequences from the Ensemb1 database. Mousetramdorin 1 genomic sequences are presented in FIG. 18. Untranslatedexon sequences are shown without spacing between the nucleotides,whereas the tramdorin open reading frame is marked by triplet nucleotidespacing.

[0317] Mouse and human tramd1 genomic sequences were compared using thePustell homology program (Pustell et al., Nucleic Acids Res 10:51(1982a)). The exons were highly conserved, but in addition, shortstretches of intronic and 5′ sequences were also conserved (FIG. 18);these sequences may be important for tramd1 RNA transcription orprocessing. Notably, the intron 3′ to exon 7, which is unspliced inmouse EST cDNA 1363993 and human cDNA htramd1 #l, contains several suchconserved sequences. In FIG. 18, nucleotides that are conserved betweenmouse and human in orientation and spacing relative to one another areshown in bold. Nucleotides and exons are numbered to the right. Genomicsequence for the coding region of exon 10 has not been determined, andtherefore exon 10 is listed in parentheses opposite a gap in thesequences that contains it. Restriction sites that correspond to theendpoints of subcloned fragments are shown in italics. Sequences betweennucleotides 5188 and 5563, and 14823 and 17056 are derived from theEnsemb1 mouse genome sequence database and are derived from C57BL/6; allother sequences are derived from al29/Sv BAC.

[0318] To determine if the BAC contained genomic sequences foradditional mouse tramdorin genes, plasmids that contained shotgun-clonedBAC DNA were arrayed on dot blots. Sequence analysis of clones thathybridized to radiolabelled tramd1 cDNA probes revealed that in additionto tramd1 sequences, the BAC also contained tramdorin 3 sequences (FIG.17B). Exons 2-8 were defined by homology to mouse tramd1 or humantramd3. Sequenced regions are shown below the exons; these sequenceswere obtained from the ends of the cloned fragments shown at the bottomof FIG. 17B, or from the Ensemb1 mouse genome sequence database.

[0319] Mouse tramdorin 3 genomic sequences (exons 1-11 and a possible12^(th) exon) are presented in FIG. 19. Mouse genomic sequencesencompassing the tramdorin3 gene are shown. Tramd3 exons, as defined byinclusion in 5′ and 3′RACE cDNA clones, and a PCR-amplified cDNA, areshown in bold. The initiation ATG codon, TAA stop codon and a putativeAATAAA (SEQ ID NO: 60) polyadenylation site, are underlined. Thesequence was assembled as follows: Unclipped sequences with homology tomouse tramd3 cDNA sequences were downloaded from the Ensemb1 traceserver (http://trace.ensemb1.org), and assembled using the AssemblyLIGNsoftware (Oxford Molecular). These sequences are derived from C57BL/6mice. In addition, mouse tramd3 genomic sequences from strain 129/Svwere obtained from subclones of a mouse BAC clone that was isolated onthe basis of its containing mouse tramd1 sequences. These 129/Svsequences are shown in the figure in italics. Several introns are notrepresented by complete sequence. The amount of missing sequence isestimated based on the size of the corresponding intron in humantramdorin3, with the exception of the distance between exon 11 and apossible 12 ^(th) exon that was identified on the basis of homology toRACE cDNA mtramd3R3′58#37. In this case, the distance is defined by theestimated 4 kb length of clone G10P619257, end sequences from which arehomologous to exon 11 and the 3′ end of RACE cDNA mtramd3R3′58#37. Notethat the exon numbering differs between mouse and human. Mouse exon1 ismost homologous to human exon1b. Mouse exon 8 is homologous to humanexon 10. Two putative polymorphisms between C57BL/6 and 129/Sv mice wereidentified. A stretch of 9 C residues in intron 6, marked by anasterisk, is 12 C residues in C57BL/6 mice. The W at position 5539 mayrepresent sequencing error or polymorphism; it is an A in 129Svsequence, and a T in C57BL/6 sequence. Discrepancies between cDNA andgenomic sequence may reflect polymorphism between C57BL/6, 129/Sv andSwiss Webster, the strain from which the cDNA that was used for RACE wasderived; alternatively, they may be sequencing errors. These are listedbelow:

[0320] 465 G-A in exon 1 12250 GTCA-TTTT, and 12272 G-T, in exon 11:likely genomic sequencing errors, based on comparison of the alternativetranslated sequences with human tramd3 and/or mouse tramd1

[0321] A comparison between mouse and human tramdorin 3 is presented inFIG. 20. The amino acid sequences of tramd3 are highly conserved betweenmouse and human, similarly to human and mouse tramd1 (FIG. 15).Additional comparisons between human tramdorins 1 and 2 and mouse tramd3confirm that this mouse locus is the homolog of human tramd3 (data notshown).

[0322] Because BACs typically contain approximately 100 kb of DNA, thepresence of tramd1 and tramd3 sequences in the same BAC suggests thatthese genes may reside more closely to one another in mice than they doin humans.

Example 11

[0323] This example provides putative and composite cDNAs for differenttramdorins.

[0324] A. Putative cDNA for human tramdorin 1 (SEQ ID NO: 3) and itsamino acid sequence (SEQ ID NO: 19) is shown in FIG. 10. Exons 8 and 9of this cDNA were identified by sequencing EST cDNA 3184556, whereasexons 7, 8, and part of the 3′ untranslated region were found inhtramd#1, which was isolated from a human sciatic nerve cDNA librarykindly provided by Dr. James Lupski of Baylor University. The remainingexons were identified as follows: A file that contains the sequence inthe Celera human genome scaffold GAx2HTBL3TT27e2.5-3.0 was generatedusing MacVector software (Oxford Molecular Ltd.). This sequence wasshown by BLAST searches of the Celera database to contain regions ofhomology to the mouse tramd1 cDNA, and to human tramd1 EST cDNA 3184556.30 kb chunks of this sequence, from nucleotides 1 to 150,000 wereanalyzed for homology to mouse tramd1 EST cDNA 1920302, human tramd1 ESTcDNA 3184556, human tramd2 EST cDNA 1837427, and human tramd3 EST cDNAs1733180, 2549054, and DFKZp434x1123 using the MacVector Pustell homologyprogram (Pustell et al., Nucleic Acids Res 10:51 (1982a)). Multipleregions of homology on the human genomic sequence were found, but onlythose in the correct order and orientation relative to known exons 6, 7and 8 were considered further as candidate exons. The genomic DNAflanking candidate exons was examined manually for 5′ and 3′ splicesites with close matches to the consensus human splice sites (Senapathyet al, Methods Enzymol 183:252 1990). Sequence between the splice siteswas translated in all three reading frames. If the amino acid sequencein a reading frame displayed conservation to the amino acid sequence ofmouse tramd1 or human tramd3, the region of homology was considered abona fide exon. In this manner, all ten coding exons of human tramd1were identified. Note that non-coding exons were not identified.

[0325] B. Putative cDNA for human tramdorin 2 (SEQ ID NO: 4), and thecorresponding amino acid sequence (SEQ ID NO: 20) is shown in FIG. 11.Exons 8-10 were identified by inclusion in EST cDNA 1837427. Remainingexons were identified as described above for human tramd1, or byhomology to mouse tramd2 cDNA. This cDNA contains the entire humantramd2 protein coding region, but does not contain the entire 3′untranslated region. In the figure, vertical lines mark exon boundaries;exons are labeled to the right of the sequence.

[0326] C. Composite human tramd3 cDNA and the corresponding amino acidsequence is shown in FIG. 21. Nucleotides 1-1311 are derived from ESTcDNA 2549054, whereas nucleotides 641-2057 are derived from EST cDNADFKZp434G1 123. The two cDNAs overlap between the 5′ end ofDFKZp434G1123, which is marked by a dot () at nucleotide 641, and anexon boundary between nucleotides 1311 and 1312, at which theirsequences diverge. A diagram of alternative human tramd3 cDNAs is shownin FIG. 8, and the full genomic sequence for the human tramdorin 3 geneis contained within the sequence presented in FIG. 9. The locations ofexon boundaries at which alternative cDNAs diverge are marked withvertical slashes (|). An additional alternative human tramd3 EST cDNA,1738130, possesses different 5′ and 3′ ends from EST cDNA 2549054 anddiverges from it at the exon boundaries shown between nucleotides 146and 147, and 875 and 876. Both EST cDNAs 1738130 and 2549054 diverge attheir 3′ termini from DFKZp434G1123, and encode truncated proteins. Theputative initiation ATG is shown in bold and is a good match to theKozak consensus (Kozak, Nucleic Acids Res 15:8125 (1987)). Two ATGsequences reside 5′ to the consensus start site in EST cDNA 2549054, asdoes one in EST cDNA 1738130; they do not match the Kozak consensus andtherefore are not likely to be used. The sequences of EST cDNAs 2549054and 1738130 converge at a splice site five nucleotides 5′ to theputative initiation ATG; the context for this ATG in EST cDNA 1738130(CTGACTGCCATGT) is a slightly weaker match to the Kozak consensus startsite than its context in EST cDNA 2549054. Thus it is possible thatmRNAs corresponding to EST cDNA 2549054 are translated more efficientlythan those that correspond to EST cDNA 1738130. The number of A residuesat the 3′ end of EST cDNA DFKZp434G1123 has been arbitrarily shortenedto 12.

[0327] D. Composite mouse tramdorin2 cDNA and corresponding amino acidsequence is shown in FIG. 22. Mouse tramdorin2 cDNAs were isolated froma mouse testicle cDNA library (Ambion) by 5′ and 3′ RLM-RACE (Maruyamaet al., Gene 138:171 (1994); Schaefer, Anal Biochem 227:255 (1995)),using a FirstChoice™ RACE-ready cDNA Kit (Ambion), and the followingprimers, located in exon 10:

[0328] 5′ AGGCTGTGCGGACAGTCAGGTCTA 3′ (SEQ ID NO: 61)

[0329] 5′ AGACGACATAGGGGACGATGATCTCAGC 3′ (SEQ ID NO: 62)

[0330] 5′ GCTGTACCAGTCAGTCAAGCTGAT 3′ (SEQ ID NO: 63)

[0331] Sequences from the 5′ and 3′ RACE cDNAs were used to generate thecomposite DNA sequence. Vertical bars in the nucleotide sequence denoteexon boundaries. The absence of a consensus polyadenylation site, andthe observation that the poly(A) sequences at the 3′ end are found ingenomic DNA, indicate that the 3′ RACE cDNA is not full length.

[0332] E. Composite mouse tramdorin 3 cDNA and corresponding amino acidsequence is shown in FIG. 23A. Mouse tramdorin 3 cDNAs were obtained asfollows: 5′ and 3′ RACE cDNAs were amplified from Swiss Webster mousebrain RACE-ready cDNA (Ambion) by 5′ and 3′ RLM-RACE (Maruyama et al.,Gene 138:171 (1994); Schaefer, Anal Biochem 227:255 (1995)), using aFirstChoice™ RACE-ready cDNA Kit (Ambion), and the following primers:

[0333] 5′ RACE primers:

[0334] 5′-CAGCAGGGAGAAGATGGACAACACAC-3′ (SEQ ID NO: 64)

[0335] 5′-AGCTGAGTGACGATGAGGAAGAAGTCCAC-3′ (SEQ ID NO: 65)

[0336] 3′ RACE primers:

[0337] 5′-CTATGGGGACACGGTGATGTATG 3′. (SEQ ID NO: 66)

[0338] 5′-GGGGAAGGCGCATCGTGGA 3′. (SEQ ID NO: 67)

[0339] A mouse tramd3 cDNA clone that contains sequences from exon 3 toexon 11 was obtained by PCR amplification of the same brain RACE-readycDNA using primers 5′ CCTCTCAGCCTGCTGGTGATTG 3′ (SEQ ID NO: 68) and

[0340] 5′ GGACACTACTGGGAGACACACAGG 3′. (SEQ ID NO: 69)

[0341] All cDNAs were cloned into pCRII (Invitrogen) resulting inGAATTCGGCT (SEQ ID NO: 70) sequences flanking the cDNAs. In FIGS.23A-23C, vertical lines mark exon boundaries; exons are labeled to theright of the Figure.

[0342] The composite mouse tramd 3 cDNA shown in FIG. 23A contains theentire tramd3 protein coding region, but does not contain the entire 3′untranslated region. It was made by combining sequences from 5′RACE cDNAclone mtramd3R5′50#22, a 580 bp cDNA which contains exons 1-5 and partof exon 6, and clone 61-45, a 1250 bp cDNA which was PCR amplified frommouse brain cDNA using primers that are specific for sequences in exon 3and 11.

[0343]FIG. 23 B shows mouse tramd3 3′ RACE cDNA 58#37. This cDNA mayrepresent an internally deleted tramd3 cDNA. It fuses sequences fromexon 7, shown in parentheses because its 3′ sequences are missing, toputative tramd3 3′ untranslated sequences that include a polyadenylationsite. These sequences are denoted with a question mark, because it isunknown whether they represent a separate exon, or the 3′ end of exon11.

[0344]FIG. 23C shows mouse tramd3 3′RACE cDNA 56#22. Similaralternatively spliced cDNAs that truncate the tramd reading frame havebeen found for mouse tramd1 and human trand3. These observations suggestthat such splicing events may have biological importance, perhaps bygenerating a truncated protein that could function as a dominantnegative regulator of tramd activity. This cDNA contains a splicejunction between exon 10 and an intracisternal A particle (IAP) repeatsequence (Aota et al., “Nucleotide sequence and molecular evolution ofmouse retrovirus-like IAP elements.” Gene 56:1-12 (1987); Ono,“Molecular biology of type A endogenous retrovirus” Kitasato Arch ExpMed 63: 77-90 (1990)). Due to the repetitive nature of the IAPsequences, the location of the putative tramd3 IAP sequence could not bedetermined.

Example 12

[0345] This example describes an assay to identify the mouse and humantramdorin ligands.

[0346] COS-7 cells are transfected with Lipofectin (Life Technologies,Grand Island, N.Y.), as described in Sagne et al. (2001; supra).Briefly, 1 day before transfection, 50,000 cells/well are plated in24-well dishes. The day of transfection, cells are washed once with 0.5ml of serum-free medium and then incubated 16-20 h with 250 μl ofserum-free medium containing a complex formed by 3 μl of Lipofectin and1 μg of pcDNA3 or pcDNA3-tramdorin. The tramdorins to be tested willinclude mouse tramdorin 1, mouse tramdorin 2, human tramdorin 1, humantramdorin 2 and human tramdorin 3. pcDNA3.1 is suitable for expressionof cDNAs. One milliliter of medium supplemented with FCS is added on thefollowing day, and transport assays are performed 36-48 h after thebeginning of transfection. Cells are washed twice with 0.5 ml ofKrebs-Ringer (KR) phosphate buffer (146 mM NaCl/3 mM KCl/1 mM CaCl₂/1 mMMgCl₂/10 mM KH₂PO₄/K₂HPO₄) adjusted at pH 7.5 and then incubated for 15min at 26° C. in KR buffer adjusted at pH 5.5, supplemented with 0.5-1μCi of [³H]GABA or [³H] labeled amino acids of interest and 100 μM GABA.Reaction is terminated by two washes with ice-cold KR buffer at pH 7.5.Cells are lysed in 0.1 N NaOH, and their radioactivity is measured afterneutralization by scintillation counting in Aquasol (Packard). [³H]GABA,L-[³H]glutamine and L-[³H]glutamate are available from AmershamPharmacia; L-[³H]proline and L-[³H]alanine are available from NEN.

Example 13

[0347] Transfection of Mouse Tramdorin1

[0348] A 1566 bp BglII-XmnI fragment of mouse EST cDNA 1920302 [whichdefines a 2.5 kb cDNA (SEQ ID NO: 13/GenBank Acession No.: AI 1780664],containing the entire coding region, was cloned between the BamH1 andEcoRV sites in the expression plasmid pcDNA3.1 (Invitrogen) andtransiently transfected into Cos, HeLa, and 293 cells. The cells weregrown in low-glucose Dulbecco's modified Eagle's Medium (DMEM)supplemented by 10% fetal bovine serum (FBS) and antibiotics (100 μg/mlpenicillin/streptomycin) in a humidified atmosphere containing 5% CO2 at37° C. Both Lipofectin (Gibco-BRL) and plasmid DNA were incubated inOptimen for 30 min in RT, then combined for another 15 min. The cells(approximately 80% confluent) were washed with Optimen, then incubatedwith the combined Lipofectin/DNA solution for 6 h at 37° C. After 6 hthe cells were washed once with Hank's BSS (calcium or magnesium free)and incubated for 3 days in DMEM at 37° C., then replated forimmunoblotting or immunostaining.

Example 14

[0349] Immunoblotting

[0350] Plates of confluent cells in 100 mm plates were harvested in coldDulbecco's PBS lacking calcium and magnesium (Life Technologies). Thecell pellet was lysed in ice-cold 50 mM Tris, pH 7.0, 1% SDS, and 0.017mg/ml phenylmethylsulfonyl fluoride (Sigma), followed by a briefsonication on ice with a dismembrator (Fisher Scientific). Proteinconcentration was determined using the BioRad kit (Bio-Rad Laboratories)according to manufacturers instructions. For each sample, after a 5-15min incubation in loading buffer at RT, 100 μg of protein lysate wereloaded onto a 12% SDS-polyacrylamide gel, electrophoresed, andtransferred to an Immobilon-polyvinylidene fluoride membrane (Millipore)over 1 hr, using a semidry transfer unit (Fisher). The blots wereblocked (5% powdered skim milk and 0.5% Tween-20 in Tris-bufferedsaline) overnight at 4° C. and incubated for 24 h at 4° C. in a rabbitantiserum against tramdorin1 (diluted 1:1,000). After washing inblocking solution and Tris-buffered saline containing 0.5% Tween-20,blots were visualized by enhanced chemiluminescence (Amersham) accordingto the manufacturer's protocols.

Example 15

[0351] Immunostaining

[0352] Transfected cells were plated onto plated onto 4-chamber glassslides (Nalge Nunc Int1) and incubated for 2-3 days to approximately 60%confluency. The cells were washed in PBS, and then fixed in acetone at−20° C. for 10 min, then blocked with 5% fish skin gelatin in PBScontaining 0.1% Triton for 1 hr in RT. Cells were labeled with rabbitanti-tramdorin1 (1:500), and processed as described below. Unfixed ratsciatic nerves were embedded in OCT and immediately frozen in a dryice-acetone bath. Five micron thick cryostat sections were thaw-mountedon SuperFrost Plus glass slides (Fisher Scientific) and stored at −-20°C. Teased nerve fibers were prepared from adult rat sciatic nerves, anddried on SuperFrost Plus glass slides overnight at room temperature andstored at −20° C. Sections and teased fibers were post-fixed andperneabilized by immersion in −20° C. acetone for 10 minutes, blocked atroom temperature for at least 1 hour in 5% fish skin gelatin containing0.5% Triton X100 in PBS, and incubated 16-48 hours at 4° C. with variouscombinations of primary antibodies: rabbit anti-tramdorin1 (1:500);mouse αrat MAG (clone 513, Boehringer Mannheim, 1:100); mouse αLAMP1(Developmental Hydridoma Bank 1:10). After incubating with the primaryantibodies, the slides were washed, incubated with the appropriatefluorescein- and rhodamine-conjugated donkey cross-affinity purifiedsecondary antibodies (diluted 1:100; Jackson ImmunoResearchLaboratories, West Grove, Pa.). Slides were mounted with Vectashield(Vector Laboratories, Inc., Burlingame, Calif.) and examined byepifluorescence with TRITC and FITC optics on a Leica DMR lightmicroscope and photographed with a cooled Hamamatsu camera or followedby image manipulation with Adobe Photoshop.

[0353] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described invention will be apparent to those skilledin the art without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in the art are intendedto be within the scope of the following claims.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20030157512). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

1. An isolated nucleic acid sequence selected from the group consistingof the cDNA sequence encoding mouse tramd 2 (SEQ ID NO: 1), the cDNAsequence encoding mouse tramd 3 (SEQ ID NO: 2), the cDNA sequenceencoding human tramd 1 (SEQ ID NO: 3), the cDNA sequence encoding humantramd 2 (SEQ ID NO: 4), the cDNA sequence encoding human tramd 3 (SEQ IDNO: 5), the cDNA sequence encoding rat tramd 1 (SEQ ID NO: 6), thegenomic sequence of mouse tramd 1 (SEQ ID NO: 7) and the genomicsequence of mouse tramd 3 (SEQ ID NO: 8).
 2. The RNA transcribed fromany of the sequences of claim
 1. 3. A vector comprising any of thesequences of claim
 1. 4. The vector of claim 3, wherein the vector is anexpression vector.
 5. A host cell transfected by any of the vectors ofclaim
 3. 6. The transfected host cell of claim 5, wherein said host cellis selected from the group consisting of COS-7 cells, mouse embryonicstem cells and yeast cells.
 7. A Xenopus oocyte injected with nucleicacid comprising any of the RNAs of claim
 2. 8. A method of detecting atramdL-STK4/Mrs-1/Krs-2 fusion in isolated human parathyroid adenomascomprising: (a) providing: (i) isolated tissue or cultured cells derivedfrom human parathyroid adenomas, (ii) differentially labeled tramdL andSTK4/Mrs-1/Krs-2 probes; (b) hybridizing said probes to said tissue,and; (c) detecting the hybridization pattern of said differentiallylabeled probes.
 9. A method of identifying proteins which interact withmouse tramd 1, comprising (a) providing: (i) a first recombinant vectorcomprising a portion of mouse tramd 1 in operable combination with theDNA binding domain of a transcriptional activator, such that a chimericprotein will be expressed, (ii) a population of second recombinantvectors, wherein said population comprises a library of cDNA sequencesin operable combination with the activation domain of a transcriptionalactivator, such that a population of chimeric proteins will beexpressed, and (iii) a yeast host comprising a reporter gene in operablecombination with the DNA binding sites for said transcriptionalactivator; (b) introducing said first vector and said population ofsecond vectors into said yeast host to generate a population oftransformed yeast; (c) subjecting said population of transformed toyeast to conditions such that said chimeric proteins are expressed; (d)screening said population for members which express said reporter gene,and; (e) isolating the coda fragment from said second vector frommembers of the population which express the reporter gene.